Antimicrobial compounds and compositions, and uses thereof

ABSTRACT

A method of enhancing the growth of an animal is provided. The method includes causing the animal to ingest or absorb an effective amount of one or more Fe III complex compounds, including but not limited to Fe III complexes comprising ligands bound to the iron center selected from amino acids or α-hydroxy acids, o-hydroxy benzoic acids or pyridine-2-carboxylic acids, such as ferric quinate, ferric tyrosine, ferric DOPA and ferric phenylalanine. Compounds which are structural and/or functional variants, derivatives and/or analogs of the foregoing compounds, as further described herein are also disclosed. Methods for inhibiting, reducing, or preventing biofilm formation or buildup on a surface; the treatment of, inhibition of growth of, and inhibition of colonization by, bacteria, both in biological and non-biological environments; disinfecting surfaces, potentiating the effects of antibiotics and other anti-microbial agents, and increasing the sensitivity of bacteria and other microorganisms, to anti-microbial agents are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Ser. No.62/188,183 filed Jul. 2, 2015, U.S. Ser. No. 62/171,081, filed Jun. 4,2015, U.S. Ser. No. 62/138,499, filed Mar. 26, 2015, U.S. Ser. No.62/137,630, filed Mar. 24, 2015 and U.S. Ser. No. 62/036,790, filed Aug.13, 2014, all of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present inventors have identified a class of compounds that has abroad range of antimicrobial and other activities, particularly againstbacteria, and has developed numerous uses for, and methods involving,the compounds. In one aspect, the invention is generally directed tocompositions, methods and uses for inhibiting, reducing, or preventingbiofilm formation or buildup on a surface or to removing, dispersing,reducing, or eradicating biofilm on a surface. In another embodiment,the invention is also generally relates to compositions, methods anduses for the treatment of, inhibition of growth of, and inhibition ofcolonization by, bacteria, both in biological and non-biologicalenvironments. In a further embodiment, the invention also relates tocompositions, methods and uses for disinfecting surfaces, both inbiological and non-biological environments, and products that have beencoated with, or treated by, the compounds or compositions of the presentinvention. In another embodiment, the invention also relates tocompositions, methods and uses for potentiating the effects ofantibiotics and other anti-microbial agents, and increasing thesensitivity of bacteria and other microorganisms, includingantibiotic-resistant bacteria, to antibiotics and/or otheranti-microbial agents, and also to reversing antibiotic resistance inbacteria. In yet another embodiment, the invention also relates tocompositions, methods and uses for enhancing the growth of animals andtheir efficiency of feed utilization, in particular by oraladministration of feed and/or drink compositions.

BACKGROUND OF THE INVENTION

A biofilm is an accumulation of microorganisms (bacteria, fungi, and/orprotozoa, with associated bacteriophages and other viruses) embedded ina polysaccharide matrix and adherent to solid biological or non-bioticsurfaces. Biofilms are medically important, accounting for over 80percent of hospital-acquired microbial infections in the body. Examplesinclude infections of the: oral soft tissues, teeth and dental implants;middle ear; gastrointestinal tract; urogenital tract; airway/lungtissue; eye; urinary tract prostheses; peritoneal membrane andperitoneal dialysis catheters, indwelling catheters for hemodialysis andfor chronic administration of chemotherapeutic agents (Hickmancatheters); cardiac implants such as pacemakers, prosthetic heartvalves, ventricular assist devices, and synthetic vascular grafts andstents; prostheses, internal fixation devices, percutaneous sutures; andtracheal and ventilator tubing. The microorganisms tend to be far moreresistant to antimicrobial agents and to be particularly difficult forthe host immune system to render an appropriate response. Severalbacterial pathogens have been shown to associate with, and in somecases, grow in biofilms, including Legionella pneumophila, S. aureus,Listeria monocytogenes, Campylobacter spp., E. coli O157:H7, Salmonellatyphimurium, Pseudomonas, Vibrio cholerae, S. epidermidis, E. faecalis,and Helicobacter pylori.

Biofilms are remarkably difficult to treat with antimicrobials.Antimicrobials may be readily inactivated or fail to penetrate into thebiofilm. In addition, bacteria within biofilms have increased (up to1,000-fold higher) resistance to antimicrobial compounds, even thoughthese same bacteria are sensitive to these agents if grown underplanktonic conditions.

In addition, bacteria embedded within biofilms are resistant to bothimmunological and non-specific defense mechanisms of the body. Contactwith a solid surface triggers the expression of a panel of bacterialenzymes, which catalyze the formation of sticky polysaccharides thatpromote colonization and protection. The structure of biofilms is suchthat immune responses may be directed only at those antigens found onthe outer surface of the biofilm, and antibodies and other serum orsalivary proteins often fail to penetrate into the biofilm. In addition,phagocytes are unable to effectively engulf a bacterium growing within acomplex polysaccharide matrix attached to a solid surface. This causesthe phagocyte to release large amounts of pro-inflammatory enzymes andcytokines, leading to inflammation and destruction of nearby tissues.Conventional therapy is characteristically ineffective against biofilms,as the minimum inhibitory concentration (MIC) of antimicrobial agentshas been shown to be 10 to 1000 fold greater than for planktonicorganisms (Hoiby, et al., Int J Antimicrob Agents, 35(4):322-32 (2010).

It is an object of the present invention to provide compositions andmethods for inhibiting or preventing biofilm formation or promotingbiofilm dissolution from surfaces of interest.

It is still an object of the present invention to provide methods forreducing the transmission of pathogens in biofilm.

It is a further object of the present invention to provide methods totreat antibiotic resistant bacteria.

It is yet a further object to provide compositions to improve growthperformance.

SUMMARY OF THE INVENTION

The present inventors have identified a class of compounds, as describedfurther below in Section III.A of this application, that havesurprisingly been found to provide a broad range of activity,particularly against a diverse array of bacteria. The present inventionprovides numerous uses for, and methods involving, the compounds,particularly in the formation of compositions. The present inventionalso provides compositions, articles and products comprising one or moreof the compounds, as described further below. The present invention alsoprovides products produced by the application of the numerous uses andmethods of the present invention, as well as downstream productsproduced therefrom.

In one embodiment, the present invention provides compounds as describedfurther below in Section III.A of this application and compositionscomprising one or more of the compounds, and methods and uses employingone or more of the compounds and/or compositions, for inhibiting,reducing, or preventing biofilm formation or buildup on a surface or toremoving, dispersing, reducing, or eradicating biofilm on a surface.Accordingly, compositions for inhibiting, reducing, or removing biofilmbuildup in a subject and/or on an article or other item are provided. Anexemplary compounds and composition include an effective amount of oneor more compound selected from Ferric Quinate (Fe-QA, also referred toherein as FeQ), and ferric complexes with L-tyrosine (Fe-Tyr), L-DOPA(Fe-DOPA), L-phenylalanine (Fe-Phe) and hydrates, salts, or derivativesthereof. See Formulas IX, VIII and VII, as defined further below,respectively.

The compositions are effective against biofilms produced by a wide rangeof microbial species including, without limitation, S. epidermidis, E.faecalis, E. coli, S. aureus, Campylobacter spp. H. pylori andPseudomonas, alone, or in combination.

In an embodiment, an article or product, including medical deviceshaving on the surface or dispersed therein one or more of the compoundsas described further below in Section III.A of this application, orcomposition comprising the one or more compounds, for example of FormulaIX (Fe-QA, or also referred to as FeQ), Formula VII (Fe-DOPA alsoreferred to as FeDOPA) and Formula VIII (Fe-Tyr), or Fe-Phe, areprepared to prevent or reduce the formation of a biofilm on the article,or product, such as to prevent or reduce the formation of a biofilm onthe medical device after implantation. The surface may be a biologicalsurface (such as a surface of a living human, animal or plant surface,or the surface of a dead or harvested animal or plant), or anon-biological surface including for example, plastics, polymers,biomaterials, and metals. The present invention also provides productstreated according to this embodiment.

In another embodiment, the invention provides compounds as describedfurther below in Section III.A of this application and compositionscomprising one or more of the compounds, and methods and uses employingone or more of the compounds and/or compositions, for the treatment of,inhibition of growth of, and inhibition of colonization by, bacteria,both in biological and non-biological environments.

In a further embodiment, the invention also relates to compounds andcompositions comprising one or more of the compounds, and methods anduses employing one or more of the compounds and/or compositions, fordisinfecting surfaces, both in biological and non-biologicalenvironments, and products that have been coated with, or treated by,one or more of the compounds and/or compositions of the presentinvention.

In another embodiment, the invention also relates to compounds andcompositions comprising one or more of the compounds, and methods anduses employing one or more of the compounds and/or compositions, forpotentiating the effects of one or more antibiotics, increasing thesensitivity of bacteria (including antibiotic-resistant bacteria) to oneor more antibiotics, and also to reversing antibiotic resistance inbacteria.

In yet another embodiment, the invention also relates to compounds andcompositions comprising one or more of the compounds, and methods anduses employing one or more of the compounds and/or compositions, forenhancing the growth of animals and their efficiency of feedutilization, in particular by oral administration of feed and drinkcompositions.

Also provided are methods of treating microbial infections in a subjectby inhibiting, reducing, or removing biofilm buildup in the subject andmethods for treating subjects with microbial infections that areresistant to antibiotics. One method includes administering to thesubject an effective amount of one or more compounds as describedfurther below in Section III.A of this application, including but notlimited to compounds according to Formula A or B as described therein,one or more compounds that bind to major outer membrane proteins (MOMPs)or FlaA of Campylobacter, a mimetic or synthetic human histo-blood groupantigen or a synthetic sugar. In one embodiment, the method includesadministering to the subject an effective amount of a compoundrepresented by Formula I, Formula II, Formula III, Formula IV, FormulaV, and Formula VI. In a preferred embodiment, the method includesadministering to the subject an effective amount of a compoundrepresented by Formula IX (ferric quinate, Fe-QA also designated asFeQ), Formula VII (Fe-DOPA), Formula VIII (Fe-Tyr), or Fe-Phe, andhydrates, salts, or derivatives thereof.

In another preferred embodiment, the one or more compounds as describedfurther below in Section III.A of this application, including but notlimited to one or more compounds according to Formula A or B as descriedtherein, one or more compounds of Formula I-IX or X-XIV, or compositionscomprising one or more of said compounds, may be used to cure, treat, orprevent symptoms of or associated with a variety of conditions asdescribed herein, such as arterial damage, gastritis, urinary tractinfections, biliary tract infections, pyelonephritis, cystitis, sinusinfections, ear infections, otitis media, otitis externa, leprosy,tuberculosis, conjunctivitis, bloodstream infections, benign prostatichyperplasia, chronic prostatitis, lung infections including chronic lunginfections of humans with cystic fibrosis, osteomyelitis, catheterinfections, bloodstream infections, skin infections, acne, rosacea,dental caries, periodontitis, gingivitis, nosocomial infections,arterial damage, endocarditis, periprosthetic joint infections, open orchronic wound infections, venous stasis ulcers, diabetic ulcers,arterial leg ulcers, pressure ulcers, endocarditis, pneumonia,orthopedic prosthesis and orthopedic implant infections, peritonealdialysis peritonitis, cirrhosis, and any other acute or chronicinfection that involves or is associated with a biofilm.

In a preferred embodiment for treating antibiotic- (or otherantimicrobial-) resistant microbial infections, the method includesadministering to the subject an effective amount of an antibiotic orother antimicrobial agent (which may be the antibiotic/antimicrobial towhich the microbial infection is resistant) and an effective amount ofone or more compounds as described further below in Section III.A ofthis application, including but not limited to one or more compoundsaccording to Formula A or B as described therein, or one or morecompounds represented by any of Formulae I to XIV, such as Formula I,Formula I, Formula II, Formula IV, Formula V, and Formula VI or,preferably, an effective amount of one or more compound represented byFormula IX, Formula VII and Formula VIII and hydrates, salts, orderivatives thereof. The antibiotic/antimicrobial agent may beadministered before the one or more compounds in accordance with thepresent invention, however, in a particularly preferred embodiment theantibiotic/antimicrobial agent is administered simultaneously (such asformulated in the same composition, or administered simultaneously inseparate compositions) or after the administration of the compounds ofthe present invention as described further below in Section III.A ofthis application, including but not limited to compounds represented byFormula I, Formula II, Formula III, Formula IV, Formula V, Formula VI,Formula VII, Formula VIII, and Formula IX, Formula X, Formula XI,Formula XII, Formula XIII and Formula XIV. The antibiotic/antimicrobialagent and/or the one or more compounds of the invention may also beincorporated into a medical device for delivery.

Also provided are methods for inhibiting biofilm buildup on a surface orfor reducing or removing biofilm from a surface. The method includescontacting the surface with an effective amount of one or more compoundsas described further below in Section III.A of this application,including but not limited to one or more compounds according to FormulaA or B as described therein, compounds that bind to major outer membraneproteins (MOMPs) or FlaA of Campylobacter, a mimetic or synthetic humanhisto-blood group antigen or a synthetic sugar, to inhibit or reducebiofilm buildup or to reduce or remove biofilm from the surface. In apreferred embodiment, the method includes administering to the subjectan effective amount of one or more compound selected from Fe-QA, Fe-Tyr,Fe-DOPA, and Fe-Phe, and hydrates, salts, or derivatives thereof, tointerfere with bacteria binding to the surface or each other. Thesurface to be treated may be contacted with the compounds by coating thesurface with the one or more compounds. In some embodiments, the surfaceis contacted by immersing the article to be treated in a compositioncomprising the one or more compounds of the present invention, orflushing, spraying, irrigating, or wiping the surface with a carriercontaining the one or more compounds of the present invention.

The disclosed methods and uses are useful for inhibiting biofilm buildup (or reducing or removing biofilm) produced by microbial speciesincluding S. epidermidis, E. faecalis, E. coli, S. aureus, Campylobacterspp. H. pylori and Pseudomonas, alone, or in combination, on/in asubject. The methods are thus also useful in treating disease conditionscaused by these and other microorganisms that are associated withbiofilm buildup.

With respect to surfaces, the disclosed methods and uses employing oneor more compounds as described further below in Section III.A of thisapplication, including but not limited to one or more compoundsaccording to Formula A or B as described therein, and more preferablyone or more compound selected from Fe-QA, Fe-Tyr, Fe-DOPA, and Fe-Phe,and hydrates, salts, or derivatives thereof, are useful for inhibitingbiofilm formation, dispersing biofilms and disinfecting articles,including but not limited to dental instruments, teeth, dentures, dentalretainers, dental braces including plastic braces (such as Invisalign),medical instruments, medical devices, contact lenses and lens cases,catheters, surfaces (e.g., tabletop, countertop, bathtub, tile, filters,membranes, etc.), tubing, drains, pipes including gas pipes, oil pipes,drilling pipes, fracking pipes, sewage pipes, drainage pipes, hoses,fish tanks, showers, children's toys, boat hulls, and cooling towers. Afurther embodiment of the present invention provides articles treated inaccordance with the foregoing methods and uses.

In further embodiments of the present invention, one or more compoundsas described further below in Section III.A of this application,including but not limited to one or more compounds according to FormulaA or B as described therein, and more preferably one or more compoundselected from Fe-QA, Fe-Tyr, Fe-DOPA, and Fe-Phe, and hydrates, salts,or derivatives thereof, may be used in methods to make antifoulingcoatings, liquid, spray and towelette dispersants, and wound irrigationsolutions.

In further embodiments of the present invention, one or more compoundsas described further below in Section III.A of this application,including but not limited to one or more compounds according to FormulaA or B as described therein, and more preferably one or more compoundselected from Fe-QA, Fe-Tyr, Fe-DOPA, and Fe-Phe, and hydrates, salts,or derivatives thereof, may also be used in cosmetic formulations,including skin treatments, acne treatments, toothpaste, and mouth rinseformulations. The compounds disclosed herein may also be applied to thebristles of toothbrushes, dental flosses, and the like, for oralhealthcare.

Also disclosed are compositions comprising one or more compounds asdescribed further below in Section III.A of this application, includingbut not limited to one or more compounds according to Formula A or B asdescribed therein, and more preferably one or more compound selectedfrom Fe-QA, Fe-Tyr, Fe-DOPA, and Fe-Phe, and hydrates, salts, orderivatives thereof, and methods using such compositions, for improvinggrowth performance of animal such as livestock (including poultry,cattle, sheep, swine and goats) and other animals as discussed furtherbelow in section II.A, and as feed and formula supplements for suchanimals, in place of, or in combination with, existing bacteriostatic orbactericidal or growth enhancing compounds. In a preferred embodimentthe compositions may be administered to animals, such as livestock, toincrease growth performance. The compositions may also be used todecrease mortality adjusted feed conversion ratios (MFCR). In apreferred embodiment, the method includes administering to the subjectan effective amount of a compound represented by Formula IX, Formula VIIand Formula VIII, or a hydrate, salt, or derivative thereof. In aparticularly preferred embodiment the compositions may be administeredto chicken and other animals to promote growth. Further relateddisclosure is provided in section II.A of this application, below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing biofilm formation by Enterococcusfaecalis at time T=0 in the presence of absence of differentconcentrations of Fe-QA. FIG. 1B is a bar graph showing biofilmformation by Enterococcus faecalis at time T=24 h in the presence orabsence of different concentrations of Fe-QA.

FIG. 2A is a bar graph showing biofilm formation by Staphylococcusepidermidis at time T=0 in the presence of absence of differentconcentrations of Fe-QA. FIG. 2B is a bar graph showing biofilmformation by Staphylococcus epidermidis at time T=24 h in the presenceof absence of different concentrations of Fe-QA.

FIG. 3A is a bar graph showing biofilm formation by Staphylococcusaureus at time T=0 in the presence of absence of differentconcentrations of Fe-QA. FIG. 3B is a bar graph showing biofilmformation by Staphylococcus aureus at time T=24 h in the presence ofabsence of different concentrations of Fe-QA.

FIG. 4A is a bar chart showing the binding of C. jejuni to the BgAgs(common ABO histo-blood group antigens), Le^(b) and H-II, after growingthe bacteria in a medium that has either Fe-QA at a concentration of0.34 mM or 3.4 mM. Binding is shown after one passage and four passages(4 generations) with Fe-QA included in the medium, and compared to acontrol without Fe-QA. FIG. 4B is a bar graph showing the binding of C.jejuni 11168 to BgAgs (common ABO histo-blood group antigens, Core-I,Core-II, H-I, H-II, Leb, Ley and Lex), and the inhibition of thisbinding by the Fe-QA (inhibitor).

FIG. 5A is a bar graph showing the effect of Fe-QA treatment onHelicobacter pylori attachment to human gastric tissue. FIG. 5B is aline graph showing the competitive inhibition of Le^(b) binding to H.pylori by Fe-QA as the concentration of Fe-QA is increased. The graph isa plot of the ratio of bound to free Le^(b) versus Fe-QA concentration.

FIG. 6A is a bar graph showing the coverage rate of PAO1 Pseudomonasaeruginosa on the surface of a glass slide, comparing Pseudomonas mediumonly as a control, PAO1 Pseudomonas+10 μM Fe-QA treatment, and PAO1Pseudomonas with no Fe-QA (X═Fe-QA). The graph shows that 100 μM Fe-QA(“X”) inhibits the formation of biofilm by P. aeruginosa. FIG. 6B is abar graph showing that Fe-QA inhibits formation of biofilm byUropathogenic E. coli (UPEC). The bar graph shows the coverage rate ofUPEC on the surface of a glass slide compared to a UPEC medium onlycontrol, and UPEC growing in the presence of 0.1 μM, 1 μM, 10 μM, and100 μM concentrations of Fe-QA.

FIG. 7A is a graph showing the growth rate of UPEC in the presence of100 μM Fe-QA and without Fe-QA over a period of 24 hours. RPMI-1640(bottom line); RPMI-1640+UPEC (middle line); RPMI-1640+100 μM FeQ+UPEC(top line). FIG. 7B is a graph showing the growth rate of Pseudomonasaeruginosa in the presence of 100 μM Fe-QA and without Fe-QA. RPMI-1640(bottom line); RPMI-1640+UPEC (middle line); RPMI 1640+100 μM FeQ+PAO1(top line).

FIG. 8A is a bar chart showing the planktonic growth rates of wildtypeCampylobacter jejuni and the same strain after mutation of the T268 ofMOMP. T268 of MOMP is replaced by glycine to form the MOMP-T strain.FIG. 8B is a bar chart showing the abilities of the wildtypeCampylobacter jejuni and the MOMP-T mutant to form biofilms.

FIG. 9 is a graph showing the impact of Fe-QA on the rate of growth of akanamycin resistant strain of E. coli. The groups, numbered from the topare as follows: (1) ΔsdiA-FEQ; (2) ΔsdiA-FEQ+Kan; (3) ΔsdiA-FEQ-Kan; (4)medium FEQ; (5) medium-FEQ+Kan; (6) medium-Kan. The graph shows the rateof growth of the strain in the presence of Fe-QA ((1)—upper line),kanamycin ((2)—triangles) and a combination of Fe-QA and kanamycin ((3),squares). Three baselines are shown for just the medium containingFe-QA, Fe-QA and kanamycin, and kanamycin alone.

FIG. 10A shows the impact on the growth curve of antibiotic resistantEnteropathogenic E. coli (EPEC) strain E2348/69 (genotype Wild Type EPECO17:H6) when grown in the presence of (i) gentamicin (1.25 μM) (greycircles), (ii) Fe-Tyr (100 μM) (inverted grey triangle ∇), (iii)gentamicin (1.25 μM) and Fe-Tyr (1.25 μM) (upright white triangle, Δ),and (iv) a control with no gentamicin or Fe-Tyr present (black circles).FIG. 10B shows the growth rate of Enteropathogenic E. coli (EPEC) strainE2348/69 when grown in the presence of (i) gentamicin (1.25 μM), (ii)Fe-Tyr (100 μM), (iii) gentamicin (1.25 μM) and Fe-Tyr (1.25 μM), and(iv) a control with no gentamicin or Fe-Tyr present.

FIGS. 11A-C show the impact on the growth curve of antibiotic resistantEnteropathogenic E. coli (EPEC) strain E2348/69 (genotype Wild Type EPECO17:H6) when grown in the presence of gentamicin (1.25 μM) andincreasing concentrations (10-68 μM) of Fe-QA (also known as FeQ). FIG.11D compares the growth rates of Enteropathogenic E. coli (EPEC) strainE2348/69 when grown in the presence of 10, 34, 68 and 100 μM Fe-QA. FIG.11E compares the growth rates of Enteropathogenic E. coli (EPEC) strainE2348/69 when grown in the presence of a fixed concentration ofgentamicin (1.25 μM) and increasing concentrations of Fe-QA ranging from10 to 100 μM.

FIG. 12 shows quantitatively the difference in the attachment of EPECcells to the plastic well surface in the absence and presence of FeQ bymeasurement of the optical absorbance of crystal violet that wasabsorbed by EPEC cells attached to the surface.

FIG. 13 is a graph showing the impact of growth rates of anantibiotic-resistant clinical isolate of Pseudomonas in the presence ofkanamycin, FeQ, and kanamycin plus FeQ compared to the strain grown inthe absence of kanamycin and FeQ.

FIGS. 14A-C show chemical structures of how FeQ can be conjugated to anagent that contains a reactive functional group suitable forimmobilizing FeQ, for example, on a surface. FIG. 14A shows theconjugation of FeQ to a calix [4] arene frame that contains aphotoreactive functional group. FIG. 14B shows the conjugation of FeQ toa calix [4] arene frame wherein the photo-reactive functional group ispositioned in a different location on the calix [4] arene frame comparedto the structure of FIG. 14B. FIG. 14C shows the conjugation of FeQ to acalix [4] arene frame functionalized with two thiol groups.

FIGS. 15A and B are chemical structures that illustrate how FeQ can beconjugated via a linker to a substance that binds to a surface. In bothstructures, the linker is spaced between functional groups Y′ and X′,attached to FeQ via Y′ and to hydroxyapatite (HA) via X′. The figuresdiffer in the point of attachment to the quinic acid ligand.

FIG. 16 is a graph showing that the wildtype O-glycosylated strain ofCampylobacter dominates infection of chicken colonized by a mixedpopulation of O-glycosylated and non-glycosylated (MOMP^(T268G))Campylobacter, and that the non-glycosylated bacteria is unable tocolonize in a mixed population.

FIG. 17 is a graph showing the average body weight (ABW) of chickenafter 42 days of growth. The graph compares the ABW at 42 days ofchicken challenged with Campylobacter-infected dirty litter at day 20and treated from days 0-42 with FeQ or FeTyr to (i) a standardcommercial target (of 2.979 kg) labeled “Target”, (ii) a negativecontrol (of 3.437 kg) labeled “CNC” where the chicken were notchallenged with Campylobacter-infected dirty litter, and (iii) apositive control (of 3.186 kg) labeled “CC” where the chicken werechallenged with Campylobacter-infected dirty litter. The graph showsthat birds challenged with Campylobacter-infected dirty litter havehigher ABW at 42 days compared to the positive control (labeled “CC”)when treated with (iv) FeQ at 0.22 g/L in drinking water and FeQ at 0.22g/kg in feed, labeled “FeQ(W+F)” with an ABW of 3.342 kg, (v) FeQ at0.22 g/L in drinking water, labeled “FeQ(W)” with an ABW of 3.407 kg,(vi) FeQ at 0.22 g/kg in feed, labeled “FeQ(F)” with an ABW of 3.464 kg,(vii) FeQ at 0.022 g/L in drinking water, labeled “FeQ(W)” with an ABWof 3.304 kg, and (viii) FeTyr at 0.02 g/L in drinking water, labeledFeTyr(W) with an ABW of 3.341 kg.

FIG. 18 is a graph showing the mortality adjusted feed conversion ratio(MFCR) of chicken after 42 days of growth. The graph compares the MFCRat 42 days of chicken challenged with Campylobacter-infected dirtylitter at day 20 and treated from days 0-42 with FeQ or FeTyr to (i) astandard commercial target (of 1.703) labeled “Target”, (ii) a negativecontrol (of 1.563) labeled “CNC” where the chicken were not challengedwith Campylobacter, and (iii) a positive control (of 1.679) labeled “CC”where the chicken were challenged with Campylobacter-infected dirtylitter. The graph shows that birds challenged withCampylobacter-infected dirty litter have lower MFCR at 42 days comparedto the positive control (labeled “CC”) when treated with (iv) FeQ at0.22 g/L in drinking water and FeQ at 0.22 g/kg in feed, labeled“FeQ(W+F)” with a MFCR of 1.595, (v) FeQ at 0.22 g/L in drinking water,labeled “FeQ(W)” with a MFCR of 1.560, (vi) FeQ at 0.22 g/kg in feed,labeled “FeQ(F)” with a MFCR of 1.563, (vii) FeQ at 0.022 g/L indrinking water, labeled “FeQ(W)” with a MFCR of 1.612, and (viii) FeTyrat 0.02 g/L in drinking water, labeled FeTyr(W) with a MFCR of 1.577.

FIG. 19 is a graph showing the number of Campylobacter colony formingunits per gram (cfu/g) of bird droppings at day 42. The graph comparesthe cfu/g at day 42 of chicken that were challenged withCampylobacter-infected dirty litter at day 20 and treated from days 0-42with FeQ or FeTyr to (i) a negative control labeled “CNC” (with a cfu/gof 28,000) where the chicken were not challenged withCampylobacter-infected dirty litter, and (ii) a positive control labeled“CC” (with a cfu/g of 1,280,000) where chickens were challenged withCampylobacter-infected dirty litter at day 21. The graph shows thatbirds treated with FeQ or FeTyr have lower levels of Campylobacter intheir droppings at day 42 when treated with (iii) FeQ at 0.22 g/L indrinking water and FeQ at 0.22 g/kg in feed, labeled “FeQ(W+F)” with acfu/g of 4,860, (iv) FeQ at 0.22 g/kg in feed, labeled “FeQ(F)” with acfu/g of 12,800, (v) FeQ at 0.022 g/L in drinking water, labeled“FeQ(W)” with a cfu/g of 900,000, and (vi) FeTyr at 0.02 g/L in drinkingwater, labeled FeTyr(W) with a cfu/g of 16,600.

FIG. 20 is a graph showing the average number of Campylobacter colonyforming units per gram (cfu/g) of caeca samples at day 42. The graphcompares the cfu/g at day 42 of chicken that were challenged withCampylobacter-infected dirty litter at day 20 and treated from days 0-42with FeQ or FeTyr to (i) a negative control labeled “Treatment-1” wherethe chicken were not challenged with Campylobacter-infected dirtylitter, and (ii) a positive control labeled “Treatment-2” where chickenswere challenged with Campylobacter-infected dirty litter at day 21. Thegraph shows that birds treated with FeQ or FeTyr have lower levels ofCampylobacter in their caeca at day 42 when treated with (iii) FeQ at0.22 g/L in drinking water and FeQ at 0.22 g/kg in feed, labeled“Treatment-3”, (iv) FeQ at 0.22 g/L in water, labeled “Treatment-5”, (v)FeQ at 0.22 g/kg in feed, labeled “Treatment-6”, (vi) FeQ at 0.022 g/Lin drinking water, labeled “Treatment-7”, and (vii) FeTyr at 0.02 g/L indrinking water, labeled “Treatment-8”.

FIGS. 21A-C show the impact on the growth curve of antibiotic resistantEnteropathogenic E. coli (EPEC) strain E2348/69 (genotype Wild Type EPECO17:H6) when grown in the presence of gentamicin (1.25 μM) andincreasing concentrations (130-200 μM) of Fe-DOPA.

FIG. 22 is shows quantitatively the difference in the attachment of EPECcells to the plastic well surface in the absence and presence of FeDOPA(also referred to as Fe-DOPA) by measurement of the optical absorbanceof crystal violet that was absorbed by EPEC cells attached to thesurface.

FIG. 23 shows 3 bar graphs at 24, 48 and 72 hours of the opticalabsorbance of crystal violet that was absorbed by the EPEC cells thatremained attached to the surface of the plastic well after a maturebiofilm formed by EPEC-pgA⁺⁺ was treated with FeTyr (shown as “FeY” inFIG. 23) at 100 μM, 150 μM and 200 μM compared to an untreated biofilm(labeled “Control”) in a crystal violet assay.

FIG. 24A shows the data from positive mode analysis, as an OPLS-DAscores plot. This shows a clear separation between fresh media (FM) andother spent media (SMWT; SMWTF; SMMT; SMMTF). FIG. 24B also shows thedata from the positive mode analysis, in which the fresh media (FM)results were removed from the plot. FIG. 24C contrasts from FIG. 24A inthat it shows the data from the negative mode analysis. FIG. 24Dcontrasts with FIG. 24B in that it shows the data from the negative modeanalysis.

FIGS. 25A and B show the results of Example 30, which investigateeffects upon antibiotic resistance of a laboratory strain of Psuedomonasaeruginosa (PAO1N) and a mixed population of clinical isolates (PAOMixed), when incubated in Luria-Bertani (LB) media alone, or withdifferent concentrations (34 μM, 100 μM, 200 μM and 340 μM) of FeQ orFePhe. FIG. 25A shows the results with PAO1N cultures. FIG. 25B showsthe results with PAO Mixed cultures.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Aerosol” as used herein refers to any preparation of a fine mist ofparticles, which can be in solution or a suspension, whether or not itis produced using a propellant.

The term “alkyl” refers to the radical of saturated aliphatic groups(i.e., an alkane with one hydrogen atom removed), includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In preferred embodiments, a straight chain or branched chain alkyl has30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straightchains, and C3-C30 for branched chains), preferably 20 or fewer, morepreferably 15 or fewer, most preferably 10 or fewer. Likewise, preferredcycloalkyls have 3-10 carbon atoms in their ring structure, and morepreferably have 5, 6, or 7 carbons in the ring structure. The term“alkyl” (or “lower alkyl”) as used throughout the specification,examples, and claims is intended to include both “unsubstituted alkyls”and “substituted alkyls”, the latter of which refers to alkyl moietieshaving one or more substituents replacing a hydrogen on one or morecarbons of the hydrocarbon backbone. Such substituents include, but arenot limited to, halogen, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester,a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate,phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic orheteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF3, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl”, as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O— alkyl, —O-alkenyl, and —O-alkynyl. The terms“aroxy” and “aryloxy”, as used interchangeably herein, can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined below. The alkoxy and aroxy groups can be substituted asdescribed above for alkyl.

“Aryl”, as used herein, refers to C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF3, —CN, and combinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aralkyloxy” can be represented by —O-aralkyl, wherein aralkylis as defined above.

“Biofilm” as used herein refers any group of microorganisms in whichcells stick to each other on a surface.

A “cream” is a viscous liquid or semi-solid emulsion of either the“oil-in-water” or “water-in-oil type”.

An “emulsion” is a composition containing a mixture of non-misciblecomponents homogenously blended together.

“Gel” as used herein is a colloid in which the dispersed phase hascombined with the continuous phase to produce a semisolid material, suchas jelly.

“Cleaning formulation”, as used herein, means a composition suitable forapplication to a surface for removing dirt and oils, for disinfecting,or a combination thereof. Cleaning formulations can be antibacterial,antimicrobial, or both. Cleaning formulations are suitable for use onthe human skin, when none of the components of the composition arepresent at concentrations that cause significant signs of irritationwhen applied to human skin. As used herein, “significant signs ofirritation” include erythema, redness, and/or swelling at the site ofinjection or at the site of application, necrosis at the site ofapplication, exfoliative dermatitis at the site of application, andsevere pain that prevents daily activity and/or requires medicalattention or hospitalization. Cleaning formulations can be suitable foruse in the human buccal cavity. Cleaning formulations can be suitablefor use with articles that, subsequent to exposure and optionally withresidual levels of cleaning composition present on and/or in thearticle, will then be contacted with the human skin or other part of thehuman body, such as wherein the article (e.g. a denture) will becontacted with the buccal cavity, or will be contacted with the eye(e.g. a contact lens). Cleaning formulations can be suitable for usewith foodstuffs and/or their packaging and may, for example, be suitablefor cleaning meat products and/or carcasses used in the production ofmeat products. Cleaning formulations may be suitable for cleaningequipment used in food production. Cleaning formulations may be suitablefor use in cleaning medical devices, including implantable medicaldevices. Many other types of cleaning formulations may also be providedby the present invention, further examples of which are discussed infurther sections of this application.

“Chronic wound” as used herein refers to a wound that fails to progressthrough an orderly and timely sequence of repair or a wound that doesnot respond to treatment and/or the demands of treatment are beyond thepatient's physical health, tolerance or stamina. Many wounds that arefirst considered to be acute wounds ultimately become chronic wounds dueto factors still not well understood. One significant factor is thetransition of planktonic bacteria within the wound to form a biofilm.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur, and selenium. Other heteroatoms includesilicon and arsenic.

“Inhibition” or “inhibiting” of biofilm formation as used herein refersto a decrease of biofilm associated microorganism formation and/orgrowth.

A “lotion” is a low- to medium-viscosity liquid formulation.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” means —SH; theterm “hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

“Oil” as used herein refers to a composition containing at least 95% wt.of a lipophilic substance. Examples of lipophilic substances include butare not limited to naturally occurring and synthetic oils, fats, fattyacids, lecithins, triglycerides and combinations thereof.

An “ointment” is a semisolid preparation containing an ointment base andoptionally one or more active agents.

“Parenteral administration”, as used herein, means administration by anymethod other than through the digestive tract or non-invasive topical orregional routes.

“Patient” or “subject” to be treated and/or used in accordance with anyof the aspect of the present invention as described herein refers toeither a human or non-human animal such as a primate, non-human primate,laboratory animal, farm animal, livestock, or a domestic pet. Exemplaryanimals can optionally include chickens, particularly a meat-typechicken such as broiler chicken, or an egg-laying chicken such as apullet or hen, or a breeder chicken. Also optionally included withoutlimitation are other poultry, such as a turkey, geese, quail or ducks,or livestock, such as cattle, sheep, goats or swine, alpaca, banteng,bison, camel, cat, deer, dog, donkey, gayal, guinea pig, horse, llama,mule, rabbit, reindeer, water buffalo, yak, although the skilled personwill appreciate that other animals, including zoo animals, captiveanimals, game animals, fish (include freshwater and saltwater fish,farmed fish, and ornamental fish), other marine and aquatic animals,including shellfish such as, but not limited to, oysters, mussels,clams, shrimps, prawns, lobsters, crayfish, crabs, cuttlefish, octopus,and squid, domestic animals such as cats and dogs, rodents (such asmice, rats, guinea pigs, hamsters), and horses, are also included, aswell as any other domestic, wild and farmed animal, including mammals,marine animals, amphibians, birds, reptiles, insects and otherinvertebrates.

“Pharmaceutically acceptable” as used herein refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals (such as one or more of the animal“patients” or “subjects” as discussed above) without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt”, as used herein, refers toderivatives of the compounds defined herein, wherein the parent compoundis modified by making acid or base salts thereof.

“Therapeutically effective” or “effective amount” as used herein meansthat the amount of the composition used is of sufficient quantity toameliorate one or more causes or symptoms of a condition, bacterialcolonization, disease or disorder. Such amelioration only requires areduction or alteration, not necessarily elimination. As used herein,the terms “therapeutically effective amount” “therapeutic amount” and“pharmaceutically effective amount” are synonymous. One of skill in theart can readily determine the proper therapeutic amount.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds. In the broadest sense, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, but are notlimited to, halogens, hydroxyl groups, or any other organic groupingscontaining any number of carbon atoms, preferably 1-14 carbon atoms, andoptionally include one or more heteroatoms such as oxygen, sulfur, ornitrogen grouping in linear, branched, or cyclic structural formats.Representative substituents include alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C3-C20 cyclic, substituted C3-C20cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

“Treatment”, “treating”, or “alleviating” as used in connection with adisease or infection refers to an intervention performed with theintention of altering or inhibiting the pathology of a disorder.

II. Aspects of the Invention

Although aspects of the invention are described throughout theapplication, some of the main aspects, which all make use of thecompounds of the present invention as described further in section III.Aof this application, can be summarized as:

-   -   (i) Enhancement of animal growth;    -   (ii) Potentiating the effect of antibiotics and other        antimicrobial agents, and addressing antibiotic resistance;    -   (iii) Inhibition of formation, and treatment of preformed,        biofilms; treating microbial infections reducing microbial        colonization; and disinfecting surfaces;    -   (iv) Compounds of the present invention as described in section        III.A of this application, and compositions comprising one or        more of said compounds.

These aspects, and further aspects of the present invention, and furtherembodiments of these aspects, will be discussed in more detail below.

A. Enhancement of Animal Growth

A first aspect of the present invention is based on the surprisingfinding that compounds of the present invention as described further insection III.A of this application, can be used to enhance animal growth.Numerous examples of this effect are provided in Example 18 of thepresent application, as discussed further below.

Accordingly, the first aspect of the present invention provides a methodof enhancing the growth of an animal, the method comprising causing theanimal to ingest and/or absorb an effective amount of one or morecompounds having the structure of Formula A or B, or other compounds ofthe invention as described further in section III.A of this applicationbelow.

In other words, the first aspect of the present invention also providesfor the use of one or more compounds having the structure of Formula Aor B, or other compounds of the invention as described further insection III.A of this application below, for enhancing the growth of ananimal, by causing the animal to ingest an effective amount of the oneor more compounds.

Typically, in the practice of the first aspect of the present invention,the one or more compounds will be presented directly to the animal foringestion and/or absorption. However, in one alternative optionalembodiment of the first aspect of the present invention, the animal maybe caused to ingest or absorb the one or more compounds having thestructure of Formula A or B, or other compounds of the invention asdescribed further in section III.A of this application below, byproviding the animal simultaneously, separately or sequentially withcomponents which cause the animal to form an effective amount of the oneor more compounds having the structure of Formula A or B, or othercompounds of the invention as described further in section III.A of thisapplication below, in situ. For example, the animal could be providedwith a source of ferrous sulfate and simultaneously, separately orsequentially with a source of quinic acid or salt thereof (or otherα-hydroxyacid), or could be provided with a source of ferrous sulfateand simultaneously, separately or sequentially with a source of anatural or synthetic amino acid, such as L-tyrosine, L-DOPA orL-phenylalanine.

In a preferred option of the first aspect of the present invention, theanimal to ingests and/or absorbs one or more compounds having thestructure of Formula A as described further in section III.A of thisapplication below, and in a further preferred option the one or morecompounds are selected from the group consisting of a complex of anamino acid with Fe III and a complex of an α-hydroxyacid with Fe III, orsalts and/or hydrates thereof. In particularly preferred options of thefirst aspect of the present invention, the one or more compounds may, ormay not, be selected from any one or more of the group consisting of acomplex of quinic acid with Fe III (such as a complex having thestructure of Formula IX), a complex of L-tyrosine with Fe III (such as acomplex having the structure of Formula VIII), a complex of L-DOPA withFe III (such as a complex having the structure of Formula VII), and acomplex of L-phenylalanine with Fe III. Accordingly, in one embodimentof the first aspect of the invention, a complex of L-tyrosine with FeIII (such as a complex having the structure of Formula VIII) isparticularly preferred. Optionally, the one or more compounds is not acomplex of quinic acid with Fe III (such as a complex having thestructure of Formula IX).

The animal may be caused to ingest or absorb the one or more compoundshaving the structure of Formula A or B, or other compounds of theinvention as described further in section III.A of this applicationbelow, by providing the one or more compounds (or component partsthereof to form the compound(s) in situ) by dietary means, such as in ormixed with an animal feed, as a dietary supplement, and/or in a drinkingwater. A further option, in the case of marine, aquatic, amphibious orother animals that live partially or fully in water, is to add the oneor more compounds (or component parts thereof to form the compound(s) insitu) into the water, such as by treatment of ponds containing farmedfish or crustaceans such as shrimp and crawfish. Accordingly, forexample, in a preferred embodiment, the one or more compounds may bepresented to the animal through one or more routes selected from thegroup consisting of an animal feed, an animal feed supplement, and indrinking water or by exposure to other water. It should be noted that,dependent on the solubility of the one or more compounds used, it may bebeneficial to introduce a co-solvent to solubilize to aid dissolution inwater at an effective concentration.

Accordingly, in a further embodiment of the first aspect of the presentinvention, there is provided an animal feed, animal feed supplement anda drinking water supply, each comprising one or more compounds havingthe structure of Formula A or B, or other compounds of the invention asdescribed further in section III.A of this application below. Suitableconcentrations of the one or more compounds to include in the animalfeed, animal feed supplement and a drinking water supply includeconcentrations as discussed further below.

Also provided herewith, in a further embodiment of the first aspect ofthe present invention, is a method for the production of an animal feedproduct or animal feed supplement product, the method comprising thesteps of incorporating one or more compounds having the structure ofFormula A or B, or other compounds of the invention as described furtherin section III.A of this application below, into the animal feed productor animal feed supplement product during the preparation of the feed orsupplement. The one or more compounds may be incorporated into theproduct at any stage during the production process and may, for example,be included before one or more heating steps, such a one or more heatingsteps that comprise exposing a composition comprising the one or morecompounds to a temperature of greater than 50° C., greater than 60° C.,greater than 70° C., greater than 80° C., greater than 90° C. or greaterthan 100° C., and preferably wherein the temperature exposure is in arange selected from 50-200° C., 60-150° C., 70-100° C. In someembodiments, a temperature range for a heating step may be in the rangeof 70-90° C., such as 75-88° C., 80-87° C., 81-86° C., or 82-85° C.

Optionally, in one embodiment, a suitable method for the production ofan animal feed, such as a feed for a chicken (including a broilerchicken) may include the steps of:

(a) combining nutritional and/or other dietary components (such as oneor more components selected from wheat, soy, soy oil, minerals and otheradditives) to form a grist or other mixture;

(b) heating the grist or other mixture in a heating step as describedabove, such as with steam at 85° C. for a time effective to kill anypathogens, such as Salmonella. A period of 5-10 minutes, such as 6-8minutes, is one example of an effective period at 85° C., although thetime can be adjusted dependent on the temperature used;

(c) cooling the heated mixture. Preferably the cooling is conducted at arate and under conditions effective to avoid the formation ofcondensation, since condensation can result in the growth of pathogensincluding Salmonella.

(d) optionally pressing the cooled mixture;

(e) forming feed pellets from the cooled mixture, such as by pelletizingusing an extruder that heats the feed to a suitable temperature, asdiscussed above, for example in the range of 82-85° C.;

(f) addition of heat sensitive additives, typically by spraying. Heatsensitive additives can include enzymes, which may (for example) beselected from the group consisting of phytase, xylase, beta-lactamase.

In accordance with the foregoing method for the production of an animalfeed product, the method comprising the step of incorporating one ormore compounds having the structure of Formula A or B, or othercompounds of the invention as described further in section III.A of thisapplication below, into the animal feed product at any one or morestages of the production, including during step (a), between steps (a)and (b), during step (b), between steps (b) and (c), during step (c),between steps (c) and (d), during step (d), between steps (d) and (e),during step (e), between steps (e) and (f), during step (f), or afterstep (f).

Other additives which may be included either at the time of adding theheat-sensitive additives, or at earlier stages, include one or moreadditives selected from the list consisting of creatine, amino acids(e.g. threonine) and salt.

An animal feed or animal feed supplement as described herein and usefulin the context of the first aspect of the present invention, or anyother aspect of the present invention, may either be a vegetarian ornon-vegetarian product. A vegetarian product contains no meat or fishproducts. A non-vegetarian diet may contain either, or both, fishproduct (such as fish meal) or meat product (such as meat derivatives,bone meal, etc.).

Also provided herewith, in a further embodiment of the first aspect ofthe present invention, is a method for the production of an animaldrinking water, the method comprising the addition of one or morecompounds having the structure of Formula A or B, or other compounds ofthe invention as described further in section III.A of this applicationbelow, into an animal drinking water supply. Suitable concentrations ofthe one or more compounds in a drinking water supply are as discussedbelow, and are typically in a concentration effective to produce theeffect of enhanced growth in accordance with the first aspect of thepresent invention. A determination of a suitable concentration may takeinto account the amount of drinking water consumed by the animal. Forexample, a broiler chicken in the UK (or at an equivalent temperature tothose used in the UK) typically consumes a daily amount of drinkingwater dependent on its age that can be calculated by reference to theage of the chicken in days multiplied of approximately 4-10 mL, such as5-9 ml, 6-8 mL, for example about 7.14 mL. Thus, for example, a 42 dayold broiler chicken may have a daily water consumption of 168 mL to 420mL per day, more typically around 300 mL per day ±30%, 20%, 15%, 10%,5%, 4%, 3%, 2%, or 1%. Broiler chicken reared at different temperaturesmay consume more (e.g. in southern USA, where temperatures in the summerwill be high and water consumption could be higher, particularly insheds where temperature is not controlled), or less water.

The animal may ingest or absorb an effective amount of one or morecompounds on a regular and repeated basis. For example, the animal mayingest or absorb an effective amount of one or more compounds weekly,every other day, every day, or more than once every day during theperformance of the method or use. In one option, the one or morecompounds are included in the an animal feed, an animal feed supplement,and/or in drinking water and the animal ingests the one or morecompounds when they eat and/or drink, and optionally every time they eatand/or drink. This ingestion or absorption an effective amount of one ormore compounds may continue through a period of time of the animal'sgrowth that may correspond to a period of time that is, is up to, or isat least, 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% orsubstantially 100% of the life of the animal from birth to death. Theingestion or absorption an effective amount of one or more compounds maystart on the day of the animal's birth, or at the age of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47 days, or more. After the animal starts to ingest orabsorb the one or compounds, the animal may continue to do so on aregular and repeated basis for a period of time that can be, or be upto, or at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 days, or more.

In the case of chickens, especially broiler chickens, it may bepreferred that the chickens ingest one or more compounds in accordancewith the present invention on a repeated and regular basis in a starterdiet, in a grower diet and/or in a finisher diet, as described furtherbelow. Chickens grown for other purposes, such as breeder chickensand/or egg layer chickens, typically receive diets that are different tothe broiler chicken, as discussed further in this application andstandard diets for breeder and egg layer chickens are well known tothose skilled in the art. In accordance with further embodiments of thefirst aspect of the present invention, the one or more compounds havingthe structure of Formula A or B, or other compounds of the invention asdescribed further in section III.A of this application below, isincorporated into an animal feed for a breeder chicken and/or egg layerchicken.

In an embodiment of the first aspect of the present invention, the oneor more compounds may be included in an animal feed, or in an animalfeed supplement, for the feed of commercial birds such as chickens,turkeys, pheasants, and ducks. In one option, the one or more compoundsmay be included in, or used to supplement, a poultry feeds, which can bea “complete” feed. A complete feed is designed to contain all theprotein, energy, vitamins, minerals, and other nutrients necessary forproper growth, egg production, and health of the birds. Feeding anyother ingredients, mixed with the feed or fed separately, beyond the useof a complete feed, can upset the balance of nutrients in the “complete”feed. Feeding additional grain or supplement with the complete poultryfeed is not recommended.

Chickens used in optimized commercial broiler production are typicallyfed different diets depending upon their age. For example, chickens forbroiler production may be raised using three diets. These diets aretypically called a “starter”, “grower” and “finisher”. “Pre-starter”diets are also possible.

The “starter”, “grower” and “finisher” are typically distinguished bycrude protein content, which is often provided by ingredients such assoybean meal (SBM). For example, a starter diet for a broiler chickenmay optionally contain a crude protein contents of around 22-25% byweight, such as 22%, 23%, 24% or 25%, with 23 or 25% being preferred. Ina further example, a grower diet for a broiler chicken may optionallycontain a crude protein contents of around 21-23% by weight, such as21%, 22% or 23%, with 22% being preferred. In a further example, afinisher diet for a broiler chicken may optionally contain a crudeprotein contents of around 19-23% by weight, such as 19%, 20%, 21%, 22%or 23%, with 19%, 20%, or 21% being preferred.

Additionally or alternatively, the “starter”, “grower” and “finisher”may be distinguished by metabolizable energy (ME) content, which istypically lowest for the starter diet and highest for the finisher diet,with the grower diet having a level between the two. For example, astarter diet for a broiler chicken may have an ME of about 3000 or 3025kcal/kg (±50, 40, 30, 20, 10, 5 or less kcal/kg). In a further example,a grower diet for a broiler chicken may have an ME of about 3100 or 3150kcal/kg (±50, 40, 30, 20, 10, 5 or less kcal/kg). In a further example,a grower diet for a broiler chicken may have an ME of about 3200 kcal/kg(±50, 40, 30, 20, 10, 5 or less kcal/kg).

Animal feeds, including chicken and most particularly broiler chickenfeeds, in accordance with the present invention may also typicallycontain one or more (preferably all) of the following:

Macro minerals, which include those selected from the group consistingof calcium, phosphorus, magnesium, sodium, potassium and chloride.

Trace Minerals, including zinc and/or selenium.

Added vitamins, which include those selected from the group consistingof vitamin A, nicotinic acid, pantothenic acid, pyridoxine (B6) andbiotin in maize and wheatbased feed. Additionally there is a basicrequirement of broiler chickens for vitamin E at 10-15 mg/kg. The needfor extra supplementation with vitamin E will depend on the level andtype of fat in the diet, on the level of selenium and on the presence ofpro- and antioxidants. Heat treatment of feeds can result in thedestruction of up to 20% of vitamin E. Choline may also be given in acomplete feed.

Non-nutritive feed additives may also be included. Enzymes are routinelyused in poultry feeds to improve digestibility of feed ingredients. Ingeneral, feed enzymes are available that act on carbohydrates, plantbound minerals and proteins. Non Starch Polysaccharide (NSP) enzymes areeconomically beneficial in wheat-based feeds. These enzymes will alsoallow greater flexibility in the levels of barley to be included in theration. Phytase enzymes can be used to enhance phytate phosphorusutilization. Protease enzymes can be included to act upon vegetableproducts. Carbohydrase enzymes can be added, and may provide beneficialresponses when used in maize-soya diets. When adding enzymes before heatprocessing of broiler feeds, there is the potential for a loss in enzymeactivity. This may be avoided by spraying enzymes on to the feed at theend of processing.

Medicinal and prophylactic drugs (other than the compounds as defined insection III.A. below) may be added. A wide range of medicinal products,e.g. coccidiostats and antibiotics, may be administered through thefeed. Antibiotic Growth Promoters/Digestion Enhancers can be includedand can, for example, provide a mode of action involving modification ofthe gut microflora, with consequential benefits in nutrient utilization.

Prebiotics can be added, and refer to a group of substances whichstimulate the growth of beneficial microorganisms, at the expense ofharmful, micro-organisms. Oligosaccharides form the largest group ofthese products at present.

Probiotics can be added to introduce live micro-organisms into thedigestive tract to assist the establishment of a stable and beneficialmicroflora. The objective is to provide the gut with positive,non-pathogenic micro-organisms which will then prevent colonization withpathogenic micro-organisms by competitive exclusion.

Organic Acids may be added. Organic acid products can be used to reducebacterial contamination of the feed (e.g. after heat treatment) and canalso encourage beneficial microflora to develop in the digestive tractof the bird.

Absorbents are used specifically to absorb mycotoxins. They may alsohave a beneficial effect on general bird health and nutrient absorption.There are a range of products available for use as absorbents, includingvarious clays and charcoal.

Antioxidants can provide important protection against nutrient loss inbroiler feeds. Some feed ingredients e.g. fish meal and fats, can beprotected. Vitamin premixes should be protected by an antioxidant unlessoptimum storage times and conditions are provided. Additionalantioxidants may be added to the final feed where prolonged storage orinadequate storage conditions are unavoidable.

Anti-Mold Agents can be added. For example, mold inhibitors may be addedto feed ingredients, which have become contaminated, or to finishedrations to reduce growth of fungi and production of mycotoxins.

Pelleting agents can be added, and are used to improve pellet hardness.Some examples of pellet binders are hemicellulose, bentonite and guargum.

Other products of possible use in broiler production include essentialoils, nucleotides, glucans and specialized plant extracts. In areas ofthe world where its use is permitted, formaldehyde can be used totreat/preserve feed.

Without limitation, exemplary “starter”, “grower” and “finisher” dietsinclude those shown in Example 18 of this application, below.

The starter diet with broiler chicks may be fed for about the first10-12 days (typically in the range of the first 7-14 days of life). Thisstarter diet may be followed by the grower diet, which is provided tothe broilers for almost 2 weeks (typically from the age of about 11-24days, although in any case, after the end of the use of the starterdiet). The finisher diet may be used for the remainder of the productionperiod (typically from the age of about 24, or 25, days to harvest).Some broiler houses will use more or less diets (for example 4 diets),and vary the timing of diet changes. Broilers are typically harvestedbetween 35 and 42 days, although this time can be longer or shorter. TheUK market typically harvests at day 30-35. Other countries, includingsome European countries, harvest as early as 25 days, although moretypically from 30 days onwards. Yet other countries, such as the US,typically harvest at 42-47 days. Non-broiler chickens, includingfree-range chickens, may be harvested at later ages. In the context ofthe practice of the first aspect of the present invention, any age ofharvest may be used, although most typically (e.g. in the context ofbroiler chickens) after the start of the finisher diet, and optionally(and without limitation) on any of days 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70 or beyond, such as up to or about 11 weeks, 12 weeks, 13 weeks,14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks ormore.

In some embodiments of the first aspect of the present invention,methods for the production of broiler chicken or other animals may beperformed on groups that are single sex (i.e. groups of solely female,or solely male animals), and/or may be performed on groups of mixed sex(i.e. mixed male and female) animals. For example, in the case of theproduction of broiler chickens, it may be appropriate to select and reartogether a single sex group of male cockerels, and it may be suitable toharvest the cockerels at an earlier age than female or mixed sex groups.For example, a single sex cockerel group of broiler chickens may beharvested at the age of around 30 days or, in other options, at the ageof any one or more of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, ormore days. For example, at the age of 30 days, an untreated cockerelgroup may have an average target weight of about 1.95 kg, whereas in thecase of the enhanced growth resulting from the performance of the methodof the first aspect of the present invention, it may be appropriate toharvest the cockerels at an earlier stage at the defined target weight,or to harvest at the same age and a higher average weight, or at thesame age and target weight with the use of a reduced consumption ofanimal feed due to greater feed conversion efficiency. In a furtherexample, a mixed sex group of broiler chickens may be harvested at theage of around 35 days or, in other options, at the age of any one ormore of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more days. For example, at theage of 35 days, an untreated mixed sex group may have an average targetweight of about 2.1-2.2 kg, whereas in the case of the enhanced growthresulting from the performance of the method of the first aspect of thepresent invention, it may be appropriate to harvest the mixed sex groupat an earlier stage at the defined target weight, or to harvest at thesame age and a higher average weight, or at the same age and targetweight with the use of a reduced consumption of animal feed due togreater feed conversion efficiency.

In accordance with the practice of the first aspect of the invention,for the purpose of enhancing the growth of broiler chickens, the one ormore compounds may be included in any one, two or three of the starter,grower and finisher diets. In one embodiment, the one or more compoundsmay be included in starter diet only. In another embodiment, the one ormore compounds may be included in grower diet only. In anotherembodiment, the one or more compounds may be included in finisher dietonly. In another embodiment, the one or more compounds may be includedin starter and grower diets only, but not the finisher diet. In anotherembodiment, the one or more compounds may be included in starter andfinisher diets only, but not the grower diet. In another embodiment, theone or more compounds may be included in grower and finisher diets only,but not the starter diet. In another embodiment, the one or morecompounds may be included in all of the starter, grower and finisherdiets.

In accordance with further embodiments of the first aspect of thepresent invention, the animal to be grown may be an egg-laying chicken.A typical process of rearing an egg-laying chicken can involve thebeginning of egg production at around 23 weeks of age, and slaughter ataround 60 weeks of age. The egg-laying chicken may be treated inaccordance with the first aspect of the present invention prior tobeginning egg laying, and/or during egg laying, and/or up to the time ofslaughter. Treatment may, for example, last for about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 weeks; the term “about” in that contextcan include the meaning of ±4, 3, 2, or 1 weeks of the stated value.Whereas, typically, egg laying chickens begin to lay eggs at 23 weeks ofage, by taking advantage of the enhanced growth and/or enhanced feedutilization of the first aspect of the present invention, it may beappropriate to begin egg production at an earlier age, such as at 18,19, 20, 21 or 22 weeks of age. Further, by taking advantage of theenhanced growth and/or enhanced feed utilization of the first aspect ofthe present invention, the present invention may be used to achieve aneffect (compared to an untreated control group that is reared underidentical conditions except for the application of the method of thefirst aspect of the present invention) selected from:

(a) the production with eggs of improved quality. Improved quality may,for example, be selected from size, shell quality, air cell, white andyolk. The shell quality is determined from any one or more of size,visual defects, specific gravity, color, breaking strength, percentshell (shell weight×100/egg weight), shell thickness, and ultrastructureof the egg. The improved quality may be reflected in a higher proportionof eggs being categorized as US grade A or AA (for example, the USstandard for grading eggs is discussed athttp://www.fao.org/docrep/005/y4628e/y4628e04.htm, the contents of whichare incorporated herein by reference);

(b) the production of eggs of increased size (such as at a weight thatis up to, or at least, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%or more); and/or

(c) the production of eggs in increased numbers (such as in an averagedaily amount, per group of at least 100 animal and/or when assessed overa period of at least 10 days, that is an amount that is up to, or atleast, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or more). Thesame approach can be taken with other egg-laying animals. Eggs producedby egg-laying chickens and other animals are labelled with informationto indicate the source and date/or of origin.

Also provided by the present invention, in accordance with a furtherembodiment of the first aspect, are one or more eggs, such as a box orcarton of eggs, produced by the animals (especially egg-laying chickens)that have been treated by a method according to the first aspect of thepresent invention. As indicated above, such eggs will typically carry alabel indicating their source and/or date of origin. Also provided aredownstream products, especially food products, produced from and/orcontaining eggs or parts thereof produced by the animals (especiallyegg-laying chickens) that have been treated by a method according to thefirst aspect of the present invention.

An animal feed of, or for use in, a first aspect of the presentinvention may comprise, or be supplemented with, one or more compoundsof the present invention in an amount of 0.001 to 20 g of the one ormore compounds per kg of feed, such as 0.002 to 15 g/kg, or at a levelof; up to, or at least, about 0.002 g/kg, 0.005 g/kg, 0.01 g/kg, 0.02g/kg, 0.03 g/kg, 0.04 g/kg, 0.05 g/kg, 0.1 g/kg, 0.2 g/kg, 0.3 g/kg, 0.4g/kg, 0.5 g/kg, 1 g/kg, 2 g/kg, 3 g/kg, 4 g/kg, 5 g/kg, 10 g/kg, 15 g/kgor 20 g/kg. An animal drinking water supply of, or for use in, the firstaspect of the present invention may comprise, or be supplemented with,one or more compounds of the present invention in an amount of 0.001 to20 g of the one or more compounds per L of water, such as 0.002 to 15g/L, or at a level of, up to, or at least, about 0.002 g/L, 0.005 g/L,0.01 g/L, 0.02 g/L, 0.03 g/L, 0.04 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.3g/L, 0.4 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 10 g/L, 15 g/Lor 20 g/L. The same concentrations can apply to water in which aquaticor other animals live.

Optionally, the methods and uses of the present invention are conductedsuch that, during the course of the treatment, the animal ingests and/orabsorbs a daily mean average total of FeQ (or an equivalent number ofmoles of any other one or more compounds according to Formula A or B, orother compounds of the invention as described further in section III.Aof this application below) of, of up to, or at least, about 100 μg, 500μg, 1 mg, 10 mg, 100 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.

In an additional or alternative option, the methods and uses of thepresent invention are conducted such that, during the course of thetreatment, the animal ingests and/or absorbs a total of FeQ (or anequivalent number of moles of any other one or more compounds accordingto Formula A or B, or other compounds of the invention as describedfurther in section III.A of this application below) of, of up to, or atleast, about (a) 5 mg, 10 mg, 50 mg, 100 mg, 500 mg, 1 g, 5 g, 10 g, 50g or 100 g per individual animal and/or (b) 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 10 mg 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg,800 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 13 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8g, 1.9 g, 2 g, 2.1 g, 2.2 g, 2.3 g, 2.4 g, 2.5 g, 2.6 g, 2.7 g, 2.8 g,2.9 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 20, g, 30g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g or 100 g per kg of final averagebody weight, as determined at the day of the final administration of theone or more compounds.

Accordingly, the present invention also provides animal feed, animalfeed supplements, drinking water supplies, and ponds (or other containedwater-based growth areas) for use in accordance with the presentinvention, and comprising the one or more compounds according to FormulaA or B, or other compounds of the invention as described further insection III.A of this application below, at the one of theconcentrations indicated above. Exemplary animal feeds of the presentinvention include chicken feeds, including (i) starter diets, growerdiets and/or finisher diets, particular for a meat-type chicken such asbroiler chicken, or (ii) for egg-laying chicken such as a pullet or hen,or (iii) for breeder chickens. Also included are feeds for otherpoultry, such as a turkey, geese, quail, pheasant, or ducks, orlivestock, such as cattle, sheep, goats or swine, alpaca, banteng,bison, camel, cat, deer, dog, donkey, gayal, guinea pig, horse, llama,mule, rabbit, reindeer, water buffalo, yak, although the skilled personwill appreciate that other feeds for animals, including zoo animals,captive animals, game animals, fish (include freshwater and saltwaterfish, farmed fish, and ornamental fish), other marine and aquaticanimals, including shellfish such as, but not limited to, oysters,mussels, clams, shrimps, prawns, lobsters, crayfish, crabs, cuttlefish,octopus, and squid, domestic animals such as cats and dogs, rodents(such as mice, rats, guinea pigs, hamsters), and horses, are alsoprovided, as well as any other domestic, wild and farmed animal,including mammals, marine animals, amphibians, birds, reptiles, insectsand other invertebrates.

In one embodiment, in the context of the first aspect of the presentinvention, the animal may be selected from the group consisting ofpoultry, such as a chicken, turkey, geese, quail, pheasant, or ducks, orlivestock, such as cattle, sheep, goats or swine, alpaca, banteng,bison, camel, cat, deer, dog, donkey, gayal, guinea pig, horse, llama,mule, rabbit, reindeer, water buffalo, yak, although the skilled personwill appreciate that other animals, including zoo animals, captiveanimals, game animals, fish (include freshwater and saltwater fish,farmed fish, and ornamental fish), other marine and aquatic animals(including shellfish such as, but not limited to, oysters, mussels,clams, shrimps, prawns, lobsters, crayfish, crabs, cuttlefish, octopus,and squid), domestic animals such as cats and dogs, rodents (such asmice, rats, guinea pigs, hamsters), and horses, as well as any otherdomestic, wild and farmed animal, including mammals, marine animals,amphibians, birds, reptiles, insects and other invertebrates may also betreated. In a particularly preferred embodiment, the animal is achicken, for example, a meat-type chicken such as broiler chicken, or anegg-laying chicken such as a pullet or hen, or a breeder chicken.

The method of enhancing the growth of an animal in accordance with thefirst aspect of the present invention may be practiced on multipleanimals, which may optionally be reared together and, further optionallywherein all animals reared together may be aged matched to within amonth, a week, or less, such as within 6, 5, 4, 3, 2 or 1 days of eachother.

For example, the method may be practiced on a group of up to, about, orat least, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵,2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶ or more,and all animals in the group may be optionally age matched as indicatedabove. The term “about” in this context can mean within ±50%, ±40%,±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2%, ±1% or less of the stated value.

The animals treated in accordance with the present invention may behealthy animals, for example, animals which are not infected with ordisadvantageously colonized by bacteria or other microorganisms. Inanother embodiment, the animals treated in accordance with the presentinvention may be unhealthy animals, for example, animals which areinfected with and/or disadvantageously colonized by bacteria or othermicroorganisms. An example of a disadvantageous bacterial colonizationis Campylobacter colonization in the GI tract of chickens; Campylobacteris not pathogenic and does not cause disease in the chicken itself(although of course it can lead to food poisoning if present in adownstream meat product produced from the chicken)—nevertheless, theCampylobacter colonization can be considered disadvantageous to thechicken as it reduces its ability to grow or efficiently utilize feed.As such, in one embodiment, an animal that is disadvantageouslycolonized by bacteria or other microorganisms is an animal whichdisplays a reduced rate of growth, reduced body weight, reduced weightgain, or less efficient feed conversion ratio due to the colonization,compared to a control animal that differs only in that it does not havethe colonization.

In some embodiments, the animals treated in accordance with the presentinvention may be animal that have been exposed to the litter (includingfeacal matter) of one or more other animals of the same or differentspecies. Optionally, the litter may be from unhealthy animals which, forexample, animals which are infected with and/or disadvantageouslycolonized by bacteria or other microorganisms. In one embodiment ofinterest to the present invention, the animals treated may be chickens,such as broiler chickens, and they may have been exposed to the litterof other chickens, such as dirty litter as described in the presentexamples and/or carrying one or more pathogens, such as Actinobacillus,Bordetalla, Campylobacter, Clostridium, Corynebacterium, Escherichiacoli, Globicatella, Listeria, Mycobacterium, Salmonella, Staphylococcus,and Streptococcus. As such, the animals to be treated in accordance withthe present invention may be chickens (or other animals) that areinfected and/or colonized by one or more of the foregoing pathogens.

Accordingly, in some embodiments, the methods and uses of the presentinvention may be non-therapeutic, in the sense that the animal to betreated is healthy and/or the method and use comprises the eventualslaughter of the animal. In other embodiments, the methods and uses ofthe present invention may include therapeutic benefits to the animals tobe treated.

In one embodiment, the methods and uses of enhancing the growth of ananimal in accordance with the first aspect of the present invention caninclude enhancing one or more characteristics selected from the groupconsisting of enhancing body weight or (in the case of a group ofanimals) average body weight (ABW), feed intake or (in the case of agroup of animals) average feed intake (AFD), weight gain or (in the caseof a group of animals) average weight gain (AWG), feed conversion ratio(FCR) and/or mortality adjusted feed conversion ratio (MFCR).

In one embodiment (for example, in the context of a group of chickensgrown in a pen) MFCR over a given period can be calculated as follows:MFCR=Total feed intake of period per pen/((total live weight ofpen+total weight of dead birds in pen)−total live weight of pen inprevious period)

For example for period 0 to 20 day, MFCR can be calculated as:MFCR_(0 to 20 day)=Total feed intake_(0-20 days)/((Total bodyweight_(at day 20)+mortality weight_(0-20 days))−Total bodyweight_(day 0)).

The enhancement in growth of the animal may be assessed over anyconvenient period during the animal's growth. It may, for example, beassessed from birth to a predetermined time point, such as up to about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180 or more days. The term “about” in this context can mean±5, ±4,±3, ±2, or ±1 days. It may, for example, be assessed from birth to apredetermined time point, such as up to about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the life span ofthe animal. It may, alternatively, not be measured from birth but bemeasured over a period of the animal's life lasting up to about 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180or more days. Again, the term “about” in this context can mean ±5, ±4,±3, ±2, or ±1 days. It may, alternatively, not be measured from birthbut be measured over a period of the animal's life representative ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,or 99% of the life span of the animal.

In the context of using the first aspect of the present invention toenhance the growth of broiler chickens, which are typically slaughteredat the average age of 35 days (in the EU) and 47 days (in the US),enhanced growth may be measured from birth up to the age of slaughter,or may be measured up to an earlier age, such as up to 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 36, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 days.Alternatively, the enhanced growth of broiler chickens may not bemeasured from birth but may be over another period of the broilerchicken's life lasting, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 36, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 days.

Enhanced growth can, in some embodiments, refer to an enhancement ingrowth in a subject animal compared to a control which is the same breedof animal as the subject, or an enhancement in a subject group ofanimals compared to a control group of an equivalent number of animalsof the same breed as the subject group, wherein the subject and controlare the same age or average age (ideally within a margin of error ofless than one day), wherein growth is measured over the same period oftime (ideally within a margin of error of less than one day), andwherein the subject and control are reared under the same conditions,differing only in that the subject receives one or more compounds of thepresent invention, in particular one or more compounds according toFormula A or B, or other compounds of the invention as described furtherin section III.A of this application below, whereas the control doesnot.

In the context of using the present invention to enhance the growth ofanimals, and in particular poultry, such as chickens and more preferablybroiler chickens, an enhancement in the rate of growth may constitute areduction in the MFCR of the subject by, by up to, or by at least, about0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12,0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20. The term “about” inthis context may include the meaning of ±5×10⁻³. The reduction in MFCRmay, for example, be measured between days 0 to 20, or days 20 to 42 ofthe life of the animal(s). Under current economic conditions, it can becalculated that a reduction in MFCR of 0.1 will lead to an approximatesaving in feed cost of about 4 US cents per bird over a 42 day growthperiod and/or about £10 GBP per tonne of animal feed used. It will beappreciated that these are substantial savings in an industry in whichcosts are typically controlled at a level of about 0.01 US cents perbird.

Further, in the context of using the first aspect of the presentinvention to enhance the growth of animals, and in particular poultry,such as chickens and more preferably broiler chickens, an enhancement inthe rate of growth may constitute an increase in the ABW of the subjectby, by up to, or by at least, about 10 g, 20 g, 30 g, 40 g, 50 g, 60 g,70 g, 80 g, 90 g, 100 g, 110 g, 120 g, 130 g, 140 g, 150 g, 160 g, 170g, 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g or more. Theterm “about” in this context may include the meaning of ±5 g, 4 g, 3 g,2 g or 1 g. The increase in the ABW may, for example, be measuredbetween days 0 to 20, or days 20 to 42 or the life of the animal(s). Inthe context of animals that normally (i.e. when not treated inaccordance with the present invention) have a higher ABW than the normalABW of broiler chickens (i.e. when not treated in accordance with thepresent invention), then the foregoing values may be increasedproportionately. That is, for example, in the case of an animal that hasa normal ABW 10-fold greater than the normal ABW of a broiler chicken,then the enhancement in the rate of growth provided by the presentinvention may constitute an increase in the ABW of the subject by, by upto, or by at least, about 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700g, 800 g, 900 g, 1000 g, 1100 g, 1200 g, 1300 g, 1400 g, 1500 g, 1600 g,1700 g, 1800 g, 1900 g, 2000 g, 2100 g, 2200 g, 2300 g, 2400 g, 2500 gor more, wherein the term “about” in this context may include themeaning of 50 g, 40 g, 30 g, 20 g or 10 g.

Further, in the context of using the first aspect of the presentinvention to enhance the growth of animals, and in particular poultry,such as chickens and more preferably broiler chickens, an enhancement inthe rate of growth may constitute an increase in the average weight gain(AWG) of the subject by, by up to, or by at least, about 10 g, 20 g, 30g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 110 g, 120 g, 130 g, 140g, 150 g, 160 g, 170 g, 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g,250 g, 260 g, 270 g, 280 g, 290 g, 300 g or more over a period ofgrowth, compared to a control animal or group of animals. The term“about” in this context may include the meaning of ±5 g, 4 g, 3 g, 2 gor 1 g. The increase in the AWG may, for example, be measured betweendays 0 to 20, or days 20 to 42 of the life of the animal(s), or during aperiod of time selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,36, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 days. In thecontext of animals that normally (i.e. when not treated in accordancewith the present invention) show a higher AWG than the normal AWG ofbroiler chickens (i.e. when not treated in accordance with the presentinvention), then the foregoing values may be increased proportionately.That is, for example, in the case of an animal that has a normal AWG10-fold greater than the normal AWG of a broiler chicken over anequivalent period of time, then the enhancement in the rate of growthprovided by the present invention may constitute an increase in the AWGof the subject by, by up to, or by at least, about 100 g, 200 g, 300 g,400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 1100 g, 1200 g, 1300g, 1400 g, 1500 g, 1600 g, 1700 g, 1800 g, 1900 g, 2000 g, 2100 g, 2200g, 2300 g, 2400 g, 2500 g, 2600 g, 2700 g, 2800 g, 2900 g, 3000 g ormore, wherein the term “about” in this context may include the meaningof ±50 g, 40 g, 30 g, 20 g or 10 g.

Prior to the present invention, in the US, the average age of slaughterof a broiler chicken is 47 days at an average weight of 2.6 kg; at theage of 42 days, the average weight may be around 2.5 kg, and in the EU,the average age of slaughter of a broiler chicken 35 days at an averageweight of 2.1-2.2 kg. It will be appreciated that, as a result of theenhanced growth provided by the methods and uses of the presentinvention, it will be possible to reach the target weight and harvestthe animal or animal products at an earlier stage of the animal's lifethan would be possible with a control. For example, in the context of abroiler chicken, it may be possible to slaughter the animal after havingachieved a target body weight 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more daysearlier than a control. In that context, a target body weight of abroiler chicken may be, may be up to, or may be at least, about 1000 g,1100 g, 1200 g, 1300 g, 1400 g, 1500 g, 1600 g, 1700 g, 1800 g, 1900 g,2000 g, 2100 g, 2200 g, 2300 g, 2400 g, 2500 g, 2600 g, 2700 g, 2800 g,2900 g, 3000 g, 3100 g, 3200 g, 3300 g, 3400 g, 3500 g or more. The term“about” in that context may include ±50 g, ±40 g, ±30 g, ±20 g or ±10 gof the stated value. To put it another way, the broiler chicken may beslaughtered at, or prior to, the age of 47, 46, 45, 44, 43, 42, 41, 40,39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26 or 25 days,ideally wherein it has reached a target body weight at the time ofslaughter. Thus, for example, in one embodiment of the presentinvention, the broiler chicken is reared to a target weight of about 2.6kg, and the method or use includes the step of slaughtering the animalafter having achieved a target body weight 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more days earlier than the age of 47 days. In another exemplaryembodiment, broiler chicken is reared to a target weight of about 2.5kg, and the method or use includes the step of slaughtering the animalafter having achieved a target body weight 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more days earlier than the age of 42 days. In another exemplaryembodiment, broiler chicken is reared to a target weight of about 2.2kg, and the method or use includes the step of slaughtering the animalafter having achieved a target body weight 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more days earlier than the age of 35 days.

In another embodiment, the animal is reared for the same amount of timeas the industry standard, but presents a greater body weight (such asabout, at least, or up to, 0.1%. 0.5%. 1%. 2%. 3%, 4%, 5%, 10%, 15%,20%, 25% or more) than the industry standard at the end of the rearingprocess. Thus, in the context of broiler chickens, the animal may beslaughtered at a weight of about 1000 g, 1100 g, 1200 g, 1300 g, 1400 g,1500 g, 1600 g, 1700 g, 1800 g, 1900 g, 2000 g, 2100 g, 2200 g, 2300 g,2400 g, 2500 g, 2600 g, 2700 g, 2800 g, 2900 g, 3000 g, 3100 g, 3200 g,3300 g, 3400 g, 3500 g or more, wherein at the time of slaughter bodyweight is about, at least, or up to, 0.1%. 0.5%. 1%. 2%. 3%, 4%, 5%,10%, 15%, 20%, 25% or more than the control. The term “about” as it isapplied to weight in that context may include ±50 g, ±40 g, ±30 g, ±20 gor ±10 g of the stated value.

In yet another embodiment, as a result of the effect of the enhancedgrowth provided by the methods and uses of the first aspect of thepresent invention, the animal is able to utilize animal feeds withgreater efficiency than a control. Accordingly, in another embodiment,the methods and uses of the present invention include the option ofrearing an animal to reach a target body weight using less animal feedthan is required for a control to reach the target weight. For example,it may be possible to use the present invention to rear an animal toreach the target weight using an amount of animal feed that is reducedin weight by 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 25% or more, compared to the amount of the sameanimal feed required by a control to reach the same target weight. Inthat context, a target body weight of a broiler chicken may be, may beup to, or may be at least, about 1000 g, 1100 g, 1200 g, 1300 g, 1400 g,1500 g, 1600 g, 1700 g, 1800 g, 1900 g, 2000 g, 2100 g, 2200 g, 2300 g,2400 g, 2500 g, 2600 g, 2700 g, 2800 g, 2900 g, 3000 g, 3100 g, 3200 g,3300 g, 3400 g, 3500 g or more. The term “about” in that context mayinclude ±50 g, ±40 g, ±30 g, ±20 g or ±10 g of the stated value.

For example, in the context of the industry standard for rearing abroiler chicken for 42 days, it is typical to provide each chicken withtotal of 5.2 kg of feed throughout its life (a mean average of 123.8 gof feed per day of life). In such a situation, in one embodiment, thepresent invention involves feeding the chicken a total amount of chickenfeed that is reduced from 5.2 kg, and/or reduced from a mean average of123.8 g feed per day, by 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%,2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or more, during its rearing.

Accordingly, the methods and uses of the present invention may furthercomprise the step of rearing the animal to permit enhanced growth.

A further embodiment in accordance with the first aspect of the presentinvention provides a method of preventing or reducing the colonizationof the gastrointestinal tract of an animal (such as an animal describedabove) with Campylobacter and/or other bacterial or microorganisms, bycausing the animal to ingest and/or absorb an effective amount of one ormore compounds having the structure of Formula A or B, or othercompounds of the invention as described further in section III.A of thisapplication below. In particular, it relates to reduction or preventionof colonization of the gastrointestinal tract of poultry (such as typesof a poultry as described above) with Campylobacter. It also relates touses of one or more compounds having the structure of Formula A or B, orother compounds of the invention as described further in section III.Aof this application below to prevent the bacteria from adhering to thewall of the gastrointestinal tract of animals and to treat or preventinfection by Campylobacter and/or other bacterial or microorganisms inhumans and animals.

Accordingly, in a further embodiment of the first aspect of the presentinvention, there is provided a method for disinfection of an animalcomprising administering to said animal at least one or more compoundshaving the structure of Formula A or B, or other compounds of theinvention as described further in section III.A of this applicationbelow in an effective amount to reduce the number of Campylobacterand/or other bacterial or microorganisms present in the gastrointestinaltract of said animal.

A further embodiment of the first aspect of the present invention alsoprovides a method for disinfection of an animal comprising administeringto said animal at least one or more compounds having the structure ofFormula A or B, or other compounds of the invention as described furtherin section III.A of this application below in an effective amount toprevent said Campylobacter and/or other bacterial or microorganisms fromforming a biofilm in the gastrointestinal tract of said animal or toreduce the amount of biofilm formed by Campylobacter and/or otherbacterial or microorganisms in the intestinal tract of said animal.

A further embodiment of the first aspect of the present invention alsoprovides a method for preventing or reducing transmission ofCampylobacter infection, and/or infection by other bacteria ormicroorganisms, from one animal to another, for example preventing orreducing spread of Campylobacter and/or infection by other bacteria ormicroorganism, within a flock or herd of animals, for example preventingspread of Campylobacter infection and/or infection by other bacteria ormicroorganisms, within a flock of chickens, including broiler chickens;said method comprising administering to said animals, for example saidherd or flock of animals, for example said flock of chickens, one ormore compounds having the structure of Formula A or B, or othercompounds of the invention as described further in section III.A of thisapplication below in an effective amount to prevent said Campylobacterand/or other bacteria or microorganisms, from forming a biofilm in thegastrointestinal tract of said animal or to reduce the amount of biofilmformed by Campylobacter and/or other bacteria or microorganisms, in theintestinal tract of said animal.

These methods may allow disinfection, prevention of biofilm formationand reduction of transmission of Campylobacter and/or other bacteria ormicroorganisms, between animals by preventing or reducing adherence ofCampylobacter and/or other bacteria or microorganisms, of thegastrointestinal tract of said animals. This is advantageous because thefewer Campylobacter and/or other bacteria or microorganisms, that are inthe gastrointestinal tract of an animal at the time of slaughter, thelower the risk of contamination of meat from the animal withCampylobacter and/or other bacteria or microorganisms. The fewerCampylobacter and/or other bacteria or microorganisms that are in thegastrointestinal tract of an animal the lower the chance of theCampylobacter and/or other bacteria or microorganisms, forming a biofilmin the gastrointestinal tract of the animal. The fewer Campylobacterand/or other bacteria or microorganisms, that are in thegastrointestinal tract of an animal, the lower the chance that theCampylobacter and/or other bacteria or microorganisms, will spread fromone animal to another, for example within a herd or flock of animals.

These methods may also be used to reduce the amount of colonisation ofthe gastrointestinal tract of any animal with Campylobacter and/or otherbacteria or microorganisms. It can be particularly advantageous toprovide the one or more compounds having the structure of Formula A orB, or other compounds of the invention as described further in sectionIII.A of this application below to animals that will be slaughtered forhuman consumption. Poultry includes birds that are used for humanconsumption such as chickens, geese, turkeys and ducks. It isparticularly, advantageous to use the compounds of the present inventionto reduce or prevent colonisation of the gastrointestinal tract ofpoultry, in particular chickens, and more particularly broiler chickens,egg laying chicken and/or breeder chickens, with Campylobacter and/orother bacteria or microorganisms because chickens are a leading sourceof human infection with Campylobacter.

The number of Campylobacter and/or other bacteria or microorganisms inthe gastrointestinal tracts of animals may be reduced by the methods ofthe present invention. In one embodiment the number of colony formingunits (cfu) of Campylobacter and/or other bacteria or microorganisms inthe gastrointestinal tract of an animal treated with the compounds ofthe present invention may be reduced by 50%, by 60%, by 70%, by 80%, by90% or by 100%. In one embodiment Campylobacter and/or other bacteria ormicroorganisms may be substantially eradicated from the gastrointestinaltract of animals treated by the method of the present invention.

10,000 cfu of Campylobacter are enough for successful chickencolonization. 1,000 cfu of Campylobacter are enough to infect a humanand cause disease in a human. Therefore, an effective amount of acompound of the present invention is enough of the compound to reducethe number of Campylobacter and/or other bacteria or microorganisms inthe gastrointestinal tract of an animal to a number that is unlikely tocause infection in humans, such as less than 10,000 cfu, 5,000 cfu,1,000 cfu, 500 cfu, 400 cfu, 300 cfu, 200 cfu, 100 cfu, 90 cfu, 80 cfu,70 cfu, 60 cfu, 50 cfu or less. The number of cfu of Campylobacterand/or other bacteria or microorganisms that would be ingested by ahuman if they ate meat from an infected animal may be related to thenumber of Campylobacter and/or other bacteria or microorganisms in thegastrointestinal tract of the animal at the time of slaughter but alsodepends on other factors such as the amount of contamination of the meatwith the contents of the gastrointestinal tract of the animal at thetime of slaughter.

An effective amount of the one or more compounds having the structure ofFormula A or B, or other compounds of the invention as described furtherin section III.A of this application below, in this context, may be anamount that is enough of the one or more compounds to preventcolonisation of the gastrointestinal tract of the animal withCampylobacter and/or other bacteria or microorganisms.

In one embodiment the one or more compounds having the structure ofFormula A or B, or other compounds of the invention as described furtherin section III.A of this application below may make Campylobacter and/orother bacteria or microorganisms less virulent and less capable ofinfecting humans even if the total number of Campylobacter and/or otherbacteria or microorganisms in the gastrointestinal tract does notdecrease. In this embodiment administering a compound of the presentinvention to an animal may affect the metabolism of Campylobacter and/orother bacteria or microorganisms and make them less adaptive toenvironment (for example, less motile) so that they cannot colonize thegastrointestinal tract and are less likely to be transmitted to otheranimals or to humans.

An effective amount of a one or more compounds provided to an animalshould be enough to provide the required degree of reduction ofCampylobacter and/or other bacterial or microorganism colonisation. Thismay depend on the type of compound and/or the size of the animal.

In one embodiment, the one of more compounds may be provided in ananimal feed, animal drink, or other compositions in concentration withinthe range of about 1 μM to about 1 μM, preferably greater than 10 μM, 20μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 11 μM, 120μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 250μM, 300 μM, 350 μM, 500 μM, 1 mM or more.

For example, the concentration of the one or more compounds may be:

(a) up to 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30μM;

(b) within a range selected from the group consisting of from 35 to 335μM, 40 to 300 μM, 50 to 300 μM, 50 to 250 μM, 50 to 200 μM, 60 to 300μM, 60 to 250 μM, 60 to 200 μM, 80 to 300 μM, 80 to 250 μM, 80 to 200μM, 100 to 300 μM, 100 to 250 μM, or 100 to 200 μM; or

(c) at least, or about, 345 μM, 350 μM, 360 μM, 370 μM, 380 μM, 390 μM,400 μM, 450 μM, 0.5 mM, 1 mM, 2 mM or more.

In another embodiment, the concentration may be within a range selectedfrom the group consisting of from about 1 μM to about 1 mM, or about 30μM to about 0.5 mM, or about 60 μM to about 0.3 mM.

In the case of the animal drink (such as drinking water) or othercomposition types, optionally, the concentration of the one or morecompounds in the composition may be within the range of 0.002 to 15 g/L,or at a level of, up to, or at least, about 0.002 g/L, 0.005 g/L, 0.01g/L, 0.02 g/L, 0.03 g/L, 0.04 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.3 g/L,0.4 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 10 g/L, 15 g/L or20 g/L

In another embodiment, the one of more compounds may be provided in ananimal feed, animal drink, or other composition in a unit dosageformulation, and/or at a concentration to deliver up to, or at least,about 1 ng, 10 ng, 50 ng, 100 ng, 500 ng, 1 g, 10 g, 50 g, 100 μg, 500μg, 1 mg, 10 mg, 100 mg, 500 mg, 1 g, 2 g, 3 g, 4 g, or 5 g of the oneor more compounds.

The methods and uses of the present invention may further comprise thestep of harvesting a product from the reared animal with enhancedgrowth.

The harvested product may be the body or part of the body of the animal.In that case, the harvesting process includes the step of slaughteringthe animal and optionally preparing an animal carcass or part thereof asa product, such as a meat product. Accordingly, the harvested body orpart of the body of then animal may be a non-food product, a foodproduct, or a precursor of a food product. Carcasses and parts ofcarcasses may go through a process known as rendering to be made intohuman and non-human foodstuffs, fats, and other material that can besold to make commercial products such as cosmetics, paint, cleaners,polishes, glue, soap and ink. Further such products that may befoodstuffs include but are not limited to blood, bone, including bonechar, bone meal, etc., broths and stocks created with animal fat, bone,and/or connective tissue, carmine also known as cochineal (food dye),casein (found in milk and cheese), civet oil (food flavoring additive),gelatin, isinglass (which, may, for example be used in clarification ofbeer and wine), L-cysteine (which may for example used in the productionof biscuits and bread), lard, meat (including fish, poultry, and game),and rennet (commonly used in the production of cheese). Meat and meatproducts may be of particular interest.

In one embodiment of particular interest in the context of the presentinvention, the animal is a chicken, for example, a meat-type chickensuch as broiler chicken, or an egg-laying chicken such as a pullet orhen, and the product is harvested from the reared animal. Mostpreferably, the animal is a meat-type chicken, such as broiler chicken,and the harvested product is a carcass or part of the carcass of thechicken. After slaughter to produce the carcass, it may or may not befurther processed, such as to remove one or more items selected from thegroup consisting of feathers, offal, neck skin, head, legs, and otheritems, and may produce a whole dressed carcass ready for sale as a meatproduct, or ready to send onto further processing. In one embodiment theprocessed carcass may retain the neck or neck skin, or at least 50%,60%, 70%, 80%, 90% or more thereof as determined either by length or byweight. The average weight of the neck or neck skin may be in the rangeof 15-25 g. Further processing may include performing a cut-up operationwherein the carcass is cut into individual parts, and may involvedeboning (i.e. where the bones are removed from specific parts) toproduce items like breast filets or other boneless products.

In one exemplary embodiment, a process for the slaughter and/orprocessing of a chicken may include any one or more of the followingmethodological step: (i) birds arrive at processing plant, typically inplastic crates; (ii) blue light is used to calm the birds; (iii) birdsare hung; (iv) birds enter a stun tank; (v) birds are slaughtered usinga neck bleed, optionally with a delay stand for bleeding out the birds;(vi) birds skin and/or feathers are heated, for example with water, toloosen pores holding the feathers; (vii) feathers are removed, e.g.using rubber fingers; (viii) an inspection is conducted to remove anybirds failing a quality control assessment; (ix) drill or otherimplement is used to create a hole in the carcass and remove anus; (x)removal of the intestines and other internal organs, typically via thepreviously-created hole; (xi) optionally, the production line splits forthe production of whole chickens and chicken parts; (xii) chicken partsmay be cut up using an automated process and through manual labor(workers slicing); optionally including the separate liver, kidneyand/or hearts; (xiii) the whole chicken carcass and/or chicken parts maybe directly labeled on the floor of the processing plant, ready for thegrocery store (further optionally including pricing) so the product cango directly on the store shelf.

It will be appreciated that alternative methods of stunning the bird areavailable, and can be substituted for the method indicated in theforegoing method and/or used more generally in accordance with the firstaspect of the present invention. Exemplary alternative methods ofstunning the bird include, for example, controlled atmosphere stunning,controlled atmosphere killing, Bi-phasic CO₂, and controlled slowdecompression.

Controlled atmosphere stunning (otherwise known as gas stunning) can beapplied to birds in transport crates, which may be conveyed through atunnel or other chamber filled with increasing concentrations of carbondioxide, inert gases (argon or nitrogen), or a mixture of these gases.The gas or gases induce unconsciousness, before slaughter. For example,at that point, the birds are hung on shackles, while insensible, andconveyed to the killing machine for slaughter.

Controlled atmosphere killing (CAK) can be operated by exposing birds tolethal concentrations of gases long enough that they are actuallykilled, rather than stunned (to avoid the risk that birds regainingconsciousness after exiting the gaseous atmosphere). For example, carbondioxide depresses the central nervous systems directly and producesrapid unconsciousness. However, carbon dioxide is aversive to chickens(usually if levels are above 20%). Inhalation of the inert gases (e.g.argon and nitrogen) can also be used, when inhaled in highconcentrations, to cause oxygen deprivation in the body, leading todeath.

Bi-phasic CO₂ is a newer gas stunning method which uses carbon dioxidein two phases to kill poultry. The first phase containing up to 40% ofcarbon dioxide (only moderately aversive to chickens), renders the birdsunconscious, the second phase follows with lethal carbon dioxide levels.

Controlled slow decompression can include the use of a Low AtmosphericPressure System (LAPS). Killing by LASP mimics the physiological effectsof ascending to high altitudes by using controlled slow decompression,which allows the body of the bird to adjust to changes in pressure andthus lose consciousness (from a lack of oxygen) with minimal discomfort.

Alternatively, the bird may not be stunned prior to slaughter, e.g. inthe case of the production of a meat product in accordance withreligious laws, such as Halal, Qurrbani/Udhia, and/or Shechita slaughterlaws.

The processing of the carcass may be conducted at adequately lowrefrigeration temperatures, such as around 1, 2, 3, 4 or 5° C.

Accordingly, following the processing of the animal carcass and/or theproduction of parts thereof, the carcass or part thereof may be furtherprocessed to produce a value added product, and this may include one ormore steps required to prepare a consumer-ready product, which mayinclude the addition of any one or more of seasoning, breading, sauces,and marinating, as well as special packaging to meet market demands forconvenient products.

Additionally, or alternatively, the harvested product may, for example,be a by-product of the animal, such as milk, eggs, wool, hair, feathers,or litter or other feacal matter and can be collected from the animalwithout the need to slaughter the animal. Such harvested products maythen be further processed and converted into other products. Forexample, in the context of milk, then further dairy products can beproduced (such as butter, cheese, curd, yoghurt, whey, milk powder, sourcream, dips and other cultured dairy foods, frozen desserts such as icecream cakes other frozen desserts made with dairy ingredients). In thecontext of eggs, then further products (in particular food products)containing or produced with the whole or part of the collected eggs canbe produced. In the context of wool, hair or feathers, then it may, forexample, be possible to produce fibers or fabrics, products containingwool, hair or feathers (such as, stuffed products), or products may bechemical or enzymatic processing of the wool, hair or feathers. Forexample, amino acids can be produced as a degradation product from wool,hair or feathers. Chicken litter can include a mixture of feces, wastedfeeds, bedding materials, and feathers can be recycled or composted andthen spread on arable land as a low cost organic fertilizer.

Any and all steps within the entire process of animal rearing, animalharvesting, animal slaughter, carcass processes, animal productproduction, food production, wrapping, labelling, shipping, stocking andselling in accordance with the first aspect of the present invention maybenefit from the application of a surface disinfection or coating inaccordance with the third aspect of the present invention, as discussedfurther below. For example, areas for rearing animals in accordance withthe first aspect of the present invention may contain one or moredisinfected surfaces achieved using the methods, uses and compositionsof the third aspect of the present invention. Containers fortransporting animals in accordance with the practice of the first aspectof the present invention may contain one or more disinfected surfacesachieved using the methods, uses and compositions of the third aspect ofthe present invention. Apparatus used in the slaughter of animals inaccordance with the practice of the first aspect of the presentinvention may contain one or more disinfected surfaces achieved usingthe methods, uses and compositions of the third aspect of the presentinvention. Apparatus used in the processing and/or labelling of ananimal carcass, or a part thereof, in accordance with the practice ofthe first aspect of the present invention may possess one or moredisinfected surfaces achieved using the methods, uses and compositionsof the third aspect of the present invention. The animal product,including a carcass, a meat product, or any other animal product asproduced in accordance with the first aspect of the present inventionmay be disinfected using the methods, uses and compositions of the thirdaspect of the present invention. Packing, containers and/or wrapping forcontaining an animal product, including a carcass, a meat product, orany other animal product as produced in accordance with the first aspectof the present invention may be disinfected using the methods, uses andcompositions of the third aspect of the present invention. Thesecombinations of the approaches set forth by the first and third aspectsof the present invention all form optional embodiments of the firstaspect of the present invention.

The present invention also provides products produced by, and/orharvested from, animals treated in accordance with the first aspect ofthe present invention, including any and all products discussed above,and downstream products including or produced therefrom.

For example, the present application provides a meat or meat productproduced in accordance with the present invention. For example, it canprovide a carcass or part thereof that is of a greater weight than astandard carcass or part thereof, or is from an animal that is youngerthan a control. Additionally, or alternatively, carcass or part thereof,or any other product obtained from the animal may have a reduced levelof microbial (such as bacterial, including Campylobacter) infection orcolonization and/or a reduced incidence of biofilms therein, compared toa control.

It will be appreciated that the foregoing methods and uses for enhancingthe growth of an animal may also be applied to humans, for example toincrease the growth of humans (such as an aid to developing body mass)and/or improve the efficiency or FCR with which humans digest food. Thiscould, for example, have applications for military personnel in helpingto reduce the burden of carrying food and/or assist in the instance offood shortages by increasing the dietary benefit of the available food.

B. Potentiating the Effect of Antibiotics and Other AntimicrobialAgents, and Addressing Antibiotic Resistance

It has been discovered that the compounds having the structure ofFormula A or B, or other compounds of the present invention as describedfurther in section III.A of this application, are particularly useful intreating or preventing infection by antibiotic-resistant microorganisms.The compounds may be administered in order to cause microorganisms tolose their resistance to antibiotics.

In Example 9, it was shown that a kanamycin-resistant strain of E. colifailed to grow when it was treated with Fe-QA and kanamycin. Yetadministration of Fe-QA alone had no impact on the growth of the strain.In Example 10, it was shown that the growth of anotherkanamycin-resistant bacterial strain, Campylobacter, was retarded whenit was treated with Fe-QA and kanamycin. In Example 14, it was shownthat the growth of an antibiotic-resistant clinical isolate ofPseudomonas was also retarded when treated with Fe-QA (also known asFeQ) and kanamycin. The effect therefore is not limited just to thebacterium, E. coli. In Example 12, it has also been shown that a wildtype strain of Enteropathogenic E. coli (EPEC) resistant to gentamicinloses its resistance when treated with a combination of Fe-QA andgentamicin. The example demonstrates that the effect is not limited tokanamycin, but is seen with other antibiotics. Furthermore, the effectis not limited to the compound, Fe-QA, but is also seen with the othercompounds. In Example 11, it has been shown that the same wild typestrain of Enteropathogenic E. coli (EPEC) resistant to gentamicin alsoloses its resistance when treated with a combination of Fe-Tyr andgentamicin. Thus the compounds are capable of causingantibiotic-resistant bacteria to lose their resistance, and thereforeadministering the compounds and antibiotics can be used to treatantibiotic resistant microorganisms (or prevent infection by thesemicroorganisms).

Accordingly, a second aspect of the present invention is based on thesurprising finding that compounds having the structure of Formula A orB, or other compounds of the present invention as described further insection III.A of this application, can be used to increase thesensitivity of microorganism to antimicrobial agents, to potentiate theeffect of antibiotics and other antimicrobial agents, and to addressantimicrobial and antibiotic resistance.

In a further preferred option of the second aspect of the presentinvention the one or more compounds are selected from the groupconsisting of a complex of an amino acid with Fe III, and a complex ofan α-hydroxyacid with Fe III, or salts and/or hydrates thereof. Inparticularly preferred options of the second aspect of the presentinvention, the one or more compounds may, or may not, be selected fromany one or more of the group consisting of a complex of quinic acid withFe III (such as a complex having the structure of Formula IX), a complexof L-tyrosine with Fe III (such as a complex having the structure ofFormula VIII), a complex of L-DOPA with Fe III (such as a complex havingthe structure of Formula VII), and a complex of L-phenylalanine with FeIII. Accordingly, in one embodiment of the second aspect of theinvention, a complex of L-tyrosine with Fe III (such as a complex havingthe structure of Formula VIII) is particularly preferred. Optionally,the one or more compounds is not a complex of quinic acid with Fe III(such as a complex having the structure of Formula IX).

In a particularly preferred embodiment, the compounds having thestructure of Formula A or B, or other compounds of the present inventionas described further in section III.A of this application may be used incombination with antimicrobial agents to treat or prevent infection byantibiotic resistant bacteria including Streptococcus pneumoniae,Campylobacter, Neisseria gonorrhoeae, Salmonella (includingdrug-resistant non-typhoidal Salmonella and drug-resistant Salmonellaserotype typhi), Methicillin-resistant Staphylococcus aureus (MRSA),Shigella, Vancomycin-resistant Enterococcus (VRE), Vancomycin-resistantStaphylococcus aureus (VRSA), Erythromycin-resistant Group AStreptococcus, Clindamycin-resistant Group B Streptococcus,Carbapenem-resistant Enterobacteriaceae (CRE), drug-resistanttuberculosis, Extended spectrum Enterobacteriaceae (ESBL),multidrug-resistant Acinetobacter (including MRAB), Clostridiumdifficile, Enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa, andUropathogenic E. coli (UPEC). In another preferred embodiment. Inanother embodiment, the compounds may be used in combination withantimicrobial agents to treat or prevent infection by antibioticresistant bacteria including S. epidermidis, E. faecalis, E. coli, S.aureus, Enteropathogenic Escherichia coli (EPEC), UropathogenicEscherichia coli (UPEC), Pseudomonas, Streptococcus anginosus,Salmonella, including Salmonella Enteritidis and Salmonella Typhimurium,Mycoplasma, Eimeria, Enterococci, Brachyspira, and Clostridiumperfringen. In a preferred embodiment, the compounds and antimicrobialagents may be administered as a pharmaceutical composition or feedadditive.

Antibiotic-resistant microorganisms (and other microorganisms resistantto other forms of anti-microbial agent) may be treated with the one ormore compounds and one or more antibiotics or other anti-microbialagents separately, sequentially or simultaneously. The one or morecompounds are preferably administered at the same time as the one ormore antibiotics or other anti-microbial agents, or preferably such thatthe compounds and antibiotic(s) are present at the same time. (Thecompounds and the antibiotics/anti-microbial agents may therefore alsobe administered sequentially.) As described previously, the compoundsmay also be formed in vivo. In this instance, the precursors may beadministered with the antibiotics or other anti-microbial agents. Forexample, the antibiotics or other anti-microbial agents could beadministered with ferrous sulfate and tyrosine (which form Fe-Tyr invivo) or ferrous sulfate and L-DOPA (which form Fe-DOPA in vivo), orferrous sulfate and L-phenylalanine (which form Fe-Phe in vivo).

The combinations of the compounds and antibiotic(s) or otheranti-microbial agent(s) may be used to treat many infections, including,but not limited to the following infections: acute bacterial skininfections, hospital-acquired bacterial pneumonia, ventilator-acquiredbacterial pneumonia, urinary tract infections, abdominal infections,kidney infections, gonorrhea, osteomyelitis, lung infections, andrespiratory tract infections.

The compounds may also be used in combination with antibiotics or otheranti-microbial agents to allow smaller doses of antibiotic or otheranti-microbial agents to be used to treat not only antibiotic-resistantmicroorganisms (and/or other microorganisms resistant to other forms ofanti-microbial agent), but also for the treatment of microorganisms thatare not resistant to antibiotics or other anti-microbial agents. Inother words, the compounds may allow smaller doses of antibiotic orother anti-microbial agent to be used to treat or prevent infections,and could also be administered to patients and animals prophylactically.For example, the compounds could be administered to poultryprophylactically so that a lower dose of antibiotic and/or otheranti-microbial agent was required to treat the birds in the event theybecome infected.

Accordingly, a second aspect of the present invention provides a methodfor the treatment or prophylaxis of a microbial infection orcolonization in a patient or animal, the method comprising administeringto the patient or animal a product selected from the group consisting ofa pharmaceutical or veterinary product, a medical device or a dietaryproduct, wherein the product comprises one or more compounds having thestructure of Formula A or B, or other compounds of the invention asdescribed further in section III.A of this application below, andpreferably wherein the pharmaceutical or veterinary product, medicaldevice or dietary product is administered to the patient or animalseparately, simultaneously, or sequentially with the administration ofone or more antimicrobials and/or antibiotics.

In other words, the second aspect of the present invention provides apharmaceutical or veterinary product, a medical device or a dietaryproduct, wherein the product comprises one or more compounds having thestructure of Formula A or B, or other compounds of the invention asdescribed further in section III.A of this application below, for use ina method of treatment or prophylaxis of a microbial infection orcolonization in a patient or animal, preferably wherein, in use, thepharmaceutical or veterinary product, medical device or dietary productis administered to the patient or animal separately, simultaneously, orsequentially with the administration of one or more antimicrobialsand/or antibiotics.

Likewise, the second aspect of the present invention also provides oneor more antimicrobials and/or antibiotics, for use in a method oftreatment or prophylaxis of a microbial infection or colonization in apatient or animal, preferably wherein, in use, the pharmaceutical orveterinary product, medical device or dietary product is administered tothe patient or animal separately, simultaneously, or sequentially withthe administration of a pharmaceutical or veterinary product, a medicaldevice or a dietary product, wherein the product comprises one or morecompounds having the structure of Formula A or B, or other compounds ofthe invention as described further in section III.A of this applicationbelow.

The microbial infection or colonization in a patient or animal may, forexample, be pathogenic or non-pathogenic microbes. Non-pathogenicmicrobes can, for example, cause colonization of a host without causingor producing any disease or disorder of the host. The microbialinfection or colonization addressed by the second aspect of the presentinvention may be prokaryotic. Examples of prokaryotic microbes includebacteria and archaea. The microbial infection or colonization addressedby the second aspect of the present invention may be eukaryotic.Examples of eukaryotic microbes include protists (such as algae, andslime-molds), fungi, multicellular micro-animals and plants includinggreen algaes.

One class of microbes of particular interest for the application of thesecond aspect of the present invention is bacteria, including pathogenicand non-pathogenic bacteria. By way of various non-limiting examples,bacteria of particular interest for the application of the second aspectof the present invention include gram positive bacteria, gram negativebacteria, biofilm-forming bacteria, extracellular bacteria,intracellular bacteria (including facultative and obligate intracellularbacteria), aerobic bacteria, and anaerobic bacteria. Some bacterialgenera of interest, without limitation, include Bacillus, Bartonella,Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia andChlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia,Francisella, Haemophilus, Helicobacter, Legionella, Leptospira,Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema,Ureaplasma, Vibrio, and Yersinia. Some bacterial species of interest,without limitation, include Bacillus anthracis, Bacillus cereus,Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borreliaburgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis,Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis,Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis,Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria,Enterococcus faecalis, Enterococcus faecium, Escherichia coli,Francisella tularensis, Haemophilus influenza, Helicobacter pylori,Legionella pneumophila, Leptospira interrogans, Leptospira santarosai,Leptospira weilii, Leptospira noguchii, Listeria monocytogenes,Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacteriumulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseriameningitides, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonellatyphi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcusagalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Treponemapallidum, Ureaplasma urealyticum, Vibrio cholera, Yersinia pestis,Yersinia enterocolitica, Yersinia pseudotuberculosis.

The treatment or prophylaxis of the second aspect of the presentinvention may be directed to one or more microorganism that haveresistance or increased tolerance to one or more antimicrobial agents.For example, the one or microorganisms may be, or include, one or moreantibiotic-resistant bacteria.

As such, in the embodiment in which the second aspect of the presentinvention is performed by administration separately, simultaneously, orsequentially with the administration of one or more antimicrobialsand/or antibiotics, then some or all of the one or more antimicrobialsand/or antibiotics may be antimicrobials and/or antibiotics to which themicroorganisms to be combated are resistant. To put it another way,typically the microorganisms to be combated may be those wherein, in theabsence of the product comprising one or more compounds having thestructure of Formula A or B, or other compounds of the invention asdescribed further in section III.A of this application below, the one ormore microorganisms is/are resistant to the one or more antimicrobialsand/or antibiotics administered to the patient or animal. Antimicrobialresistance can include the meaning of resistance of a microorganism toan antimicrobial drug that was originally effective for treatment ofinfections caused by it. Resistant microorganisms are able to withstandattack by antimicrobial drugs, such as antibacterial drugs (e.g.antibiotics), antifungals, antivirals, and antimalarials, so thatstandard treatments become ineffective and infections persist,increasing the risk of spread to others. The evolution of resistantstrains is a natural phenomenon that occurs when microorganismsreplicate themselves erroneously or when resistant traits are exchangedbetween them. The use and misuse of antimicrobial drugs accelerates theemergence of drug-resistant strains. Poor infection control practices,inadequate sanitary conditions and inappropriate food-handling encouragethe further spread of antimicrobial resistance.

In one embodiment of the second aspect of the present invention, themicroorganism is an antibiotic-resistant microorganism selected from thegroup consisting of a gram positive bacterium, a gram negativebacterium, a biofilm-forming bacterium, Streptococcus pneumoniae,Campylobacter, Neisseria gonorrhoeae, Salmonella (includingdrug-resistant non-typhoidal Salmonella and drug-resistant Salmonellaserotype typhi), Methicillin-resistant Staphylococcus aureus (MRSA),Shigella, Vancomycin-resistant Enterococcus (VRE), Vancomycin-resistantStaphylococcus aureus (VRSA), Erythromycin-resistant Group AStreptococcus, Clindamycin-resistant Group B Streptococcus,Carbapenem-resistant Enterobacteriaceae (CRE), drug-resistanttuberculosis, Extended spectrum Enterobacteriaceae (ESBL),multidrug-resistant Acinetobacter (including MRAB), Clostridiumdifficile, Enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa, H.pylori, Streptococcus anginosus and Uropathogenic E. coli (UPEC).

However, the practice of the second aspect of the invention is notlimited to the treatment or prophylaxis of resistant microorganisms. Thesecond aspect of the present invention can also be used to increase thesensitivity of non-resistant microorganisms to antimicrobial agents, andthereby provide for a treatment that uses lower dosages of antimicrobialagents, and/or shorter treatment durations with antimicrobial agents,and/or more effective treatment outcomes with antimicrobial agents.

Accordingly, in a further embodiment of the second aspect of the presentinvention, the method, or the product for use, is for potentiating theantimicrobial (including antibiotic) effect of the separately,simultaneously, or sequentially administered one or more antimicrobialagents (including one or more antibiotics). For example, in a furtherembodiment, the amount of the separately, simultaneously, orsequentially administered one or more antimicrobial agents (includingone or more antibiotics) may be less than a therapeutically effective ortherapeutically optimal dose of the one or more antimicrobial agents(including one or more antibiotics) when administered to the patient oranimal that is not in receipt of the product. In another embodiment, theamount of the separately, simultaneously, or sequentially administeredone or more antimicrobial agents (including one or more antibiotics) maybe 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, less than atherapeutically effective or therapeutically optimal dose of the one ormore antibiotics when administered to the patient or animal that is notin receipt of the product. In another embodiment, the treatment durationof the patient receiving the treatment or prophylaxis of the secondembodiment may be 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% ormore, less than the treatment duration required when the patient oranimal is not in receipt of the product.

Preferably, the subject to be treated in accordance with any embodimentof the second aspect of the present invention, is a human patient. Thehuman may be a male. Alternatively the human may be a female. The humanmay be aged up to, or greater than, 1 month, 2, months, 3 months, 4months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years,6 years, 7 years, 8 years 9 years, 10 years, 15 years 20 years, 30,years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, 100years or more.

Alternatively, subject to be treated in accordance with any embodimentof the second aspect of the present invention, may be an animal. Withoutlimitation, animals for treatment or prophylaxis according to the secondaspect of the present invention may be selected from the groupconsisting of domestic, wild and farmed animal, including mammals,marine animals, amphibians, birds, reptiles, insects and otherinvertebrates. Without limitation, exemplary animals for treatment orprophylaxis include poultry, such as a chicken, turkey, geese, quail,pheasants, or ducks; livestock, such as cattle, sheep, goats or swine,alpaca, banteng, bison, camel, cat, deer, dog, donkey, gayal, guineapig, horse, llama, mule, rabbit, reindeer, water buffalo, yak; zooanimals; captive animals; game animals; marine or aquatic animals suchas fish (include freshwater and saltwater fish, farmed fish, andornamental fish) and shellfish including but not limited to oysters,mussels, clams, shrimps, prawns, lobsters, crayfish, crabs, cuttlefish,octopus, and squid; domestic animals, such as cat or dog, a rodent(mice, rats, guinea pigs, hamsters), horse.

The one or more antimicrobial agents used and/or referred to in thesecond aspect of the present invention include those listed anddiscussed in section III.B of this application, below. In oneembodiment, at least one, or all, of the one or more antimicrobialagents is/are an antibiotic. The one or more antibiotics may, forexample, be selected from the group consisting of aminoglycosides,ansaycins, carbapenems, cephalosporins, glycopeptides, lincosamides,lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones,penicillins, polypeptides, quinolones/fluoroquinolone, sulfonamides,tetracyclines, clofazimine, dapsone, capreomycin, cycloserine,ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin (rifampin),rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole, andtrimethoprim; and combinations thereof. More specific antibioticssuitable for use in accordance with the second aspect of the presentinvention include those listed and discussed in section III.B of thisapplication, below.

In one embodiment of the second aspect of the present invention, theproduct comprising the one or more compounds having the structure ofFormula A or B, or other compounds of the invention as described furtherin section III.A of this application below is selected from the groupconsisting of a pharmaceutical or veterinary product. In one embodiment,it is a pharmaceutical product suitable for use with humans. In anotherembodiment, it is veterinary product suitable for use with animals,including but not limited to one or more animals selected from the groupconsisting of domestic, wild and farmed animal, including mammals,marine animals, amphibians, birds, reptiles, insects and otherinvertebrates. Without limitation, exemplary animals for treatment orprophylaxis include poultry, such as a chicken, turkey, geese, quail,pheasant, or ducks; livestock, such as cattle, sheep, goats or swine,alpaca, banteng, bison, camel, cat, deer, dog, donkey, gayal, guineapig, horse, llama, mule, rabbit, reindeer, water buffalo, yak; zooanimals, captive animals, game animals; marine or aquatic animals suchas fish (include freshwater and saltwater fish, farmed fish, andornamental fish) and shellfish including but not limited to oysters,mussels, clams, shrimps, prawns, lobsters, crayfish, crabs, cuttlefish,octopus, and squid; domestic animals, such as cat or dog, a rodent(mice, rats, guinea pigs, hamsters), horse. In a preferred embodiment,the animal is a chicken, for example, a meat-type chicken such asbroiler chicken, or an egg-laying chicken such as a pullet or hen, or abreeder chicken. The pharmaceutical or veterinary product may or may notadditionally include the one or more antimicrobial agents (in theembodiment that it does not, then in accordance with the second aspectof the present invention, the product and microbial agent are intendedto be administered to the subject in separate compositions, eitherseparately, simultaneously or sequentially). The pharmaceutical orveterinary product may include one or more excipients, such as discussedin section III.C of this application, below. The pharmaceutical orveterinary product may be presented as a parenteral formulation, such asdiscussed below in section III.C.1 of this application, including acontrolled release formulation, such as discussed below in sectionIII.C.1(a) of this application, and injectable or implantableformulation, such as discussed below in section III.C.1(b) of thisapplication. The pharmaceutical or veterinary product may be presentedas a enteral formulation, such as discussed below in section III.C.2 ofthis application, including a controlled release enteral formulation,such as discussed below in section III.C.2(a) of this application, withfurther reference to extended release dosage forms and delayed releasedosage forms as discussed therein. The pharmaceutical or veterinaryproduct may be presented as a topical formulation, such as discussedbelow in section III.C.3 of this application, including as an emulsion,lotion, cream, ointment, gel, or foam as discussed in parts (a), (b),(c), (d) (e) and (f) respectively below in section III.C.3 of thisapplication.

In another embodiment, the product comprising the one or more compoundshaving the structure of Formula A or B, or other compounds of theinvention as described further in section III.A of this applicationbelow is a medical device. The device may or may not additionallyinclude the one or more antimicrobial agents (in the embodiment that itdoes not, then in accordance with the second aspect of the presentinvention, the device and microbial agent are intended to beadministered to the subject in separate compositions, either separately,simultaneously or sequentially). Medical devices that can comprise theone or more compounds as defined in section III.A of this applicationcan include, without limitation, wound dressings or medical implants.Further examples include tubing and other surface medical devices, suchas urinary catheter, stents, mucous extraction catheter, suctioncatheter, umbilical cannula, contact lenses, intrauterine devices,intravaginal and intraintestinal devices, endotracheal tubes,bronchoscopes, dental prostheses and orthodontic devices, surgicalinstruments, dental instruments, tubing, dental water lines, dentaldrain tubes, fabrics, paper, indicator strips (e.g., paper indicatorstrips or plastic indicator strips), adhesives (e.g., hydrogeladhesives, hot-melt adhesives, or solvent-based adhesives), bandages,tissue dressings or healing devices and occlusive patches, and any othersurface devices used in the medical field. Devices may includeelectrodes, external prostheses, fixation tapes, compression bandages,and monitors of various types. Medical devices also include any devicethat may be placed at the insertion or implantation site such as theskin near the insertion or implantation site, and which include at leastone surface which is susceptible to colonization by biofilm embeddedmicroorganisms. In one specific embodiment, a composition is integratedinto an adhesive, such as tape, thereby providing an adhesive, which canpresent and/or deliver the one or more compounds on at least one surfaceof the adhesive. In a particularly preferred embodiment the followingdevices may comprise, include and/or be coated with the compounds:catheters, including central venous catheters, urinary catheters,dialysis catheters, and indwelling catheters (for example, catheters forhemodialysis and for administration of chemotherapeutic agents), cardiacimplants including mechanical heart valves, stents, ventricular assistdevices, pacemakers, cardiac rhythm management (CRM) devices, cardiacresynchronization therapy devices (CRTs), and implantable cardioverterdefibrillators (ICDs), synthetic vascular grafts, arteriovascularshunts, cerebral spinal fluid shunts, cochlear devices, prostheticjoints, orthopedic implants, internal fixation devices, bone cements,percutaneous sutures, surgical mesh and surgical patches includinghernia repair meshes and patches, breast reconstruction meshes andpatches, meshes and patches for breast and face lifts, slings, andmeshes and patches for pelvic floor reconstruction, tracheal andventilator tubing, wound dressings, biological implants (includingallografts, xenografts and autografts), penile implants, intrauterinedevices, endotracheal tubes, and contact lenses.

In another embodiment, the product comprising the one or more compoundshaving the structure of Formula A or B, or other compounds of theinvention as described further in section III.A of this applicationbelow is a dietary product. The dietary product may or may notadditionally include the one or more antimicrobial agents (in theembodiment that it does not, then in accordance with the second aspectof the present invention, the dietary product and microbial agent areintended to be administered to the subject in separate compositions,either separately, simultaneously or sequentially). Dietary products caninclude, for example, food stuffs, dietary supplements, drinks and anyother compositions taken orally, which incorporate the one or morecompounds having the structure of Formula A or B, or other compounds ofthe invention as described further in section III.A of this applicationbelow.

The one or more compounds are selected from the group consisting of acomplex of an amino acid with Fe III, and a complex of an α-hydroxyacidwith Fe III, or salts and/or hydrates thereof. The one or more compoundsmay, or may not, be selected from any one or more of the groupconsisting of a complex of quinic acid with Fe III (such as a complexhaving the structure of Formula IX), a complex of L-tyrosine with Fe III(such as a complex having the structure of Formula VIII), a complex ofL-DOPA with Fe III (such as a complex having the structure of FormulaVII), and a complex of L-phenylalanine with Fe III. Accordingly, in oneembodiment, a complex of L-tyrosine with Fe III (such as a complexhaving the structure of Formula VIII) is particularly preferred.Optionally, the one or more compounds is not a complex of quinic acidwith Fe III (such as a complex having the structure of Formula IX).

A complex of quinic acid with Fe III (Fe-QA, also denoted FeQ), such asdefined by Formula IX, can be used with any one or more of the foregoingantibiotics or other antimicrobials, either formulated together in thesame composition for administration or presented in separatecompositions for use separately, simultaneously or sequentially.

A complex of quinic acid with Fe III (Fe-QA, also denoted FeQ), such asdefined by Formula IX, can be used with any one or more of the foregoingantibiotics or antimicrobials, either formulated together in the samecomposition for administration or presented in separate compositions foruse separately, simultaneously or sequentially.

A complex of L-tyrosine with Fe III (Fe-Tyr), such as defined by FormulaVIII, can be used with any one or more of the foregoing antibiotics orantimicrobials, either formulated together in the same composition foradministration or presented in separate compositions for use separately,simultaneously or sequentially.

A complex of L-DOPA with Fe III (3,4 dihydrophenylalanine) (Fe-DOPA),such as defined by Formula VII, can be with any one or more of theforegoing antibiotics or antibiotics, either formulated together in thesame composition for administration or presented in separatecompositions for use separately, simultaneously or sequentially.

A complex of L-phenylalanine with Fe III (Fe-Phe), can be used with anyone or more of the foregoing antibiotics or antibiotics, eitherformulated together in the same composition for administration orpresented in separate compositions for use separately, simultaneously orsequentially.

In one embodiment in which the product selected from the groupconsisting of a pharmaceutical or veterinary product, a medical deviceor a dietary product comprising the combination of one or more compoundshaving the structure having the structure of Formula A or B, or othercompounds as described further in section III.A of this applicationbelow, and one or more antibiotics or other antimicrobial agents, thenoptionally the amount (in weight, or moles) and/or concentration of theone or more antibiotics and/or other antimicrobial agents in the productis less than (e.g. a reduction of up to, or at least, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 35%, 30%, 40%, 50%/a, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) in comparisonto a therapeutically effective or therapeutically optimal amount orconcentration of the one or more antibiotics and/or other antimicrobialagents when administered to the patient or animal that is not in receiptof the product.

The product may be presented in a unit dosage formulation, andoptionally the unit dosage formulation may include the one or moreantibiotics and/or other antimicrobial agents in an amount (in weight,or moles) or and/or concentration that is less than (e.g. a reduction ofup to, or at least, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,35%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more) in comparison a therapeutically effective ortherapeutically optimal dose of the one or more antibiotics whenadministered to the patient or animal that is not in receipt of theproduct.

Also provided is a product per se, such as a pharmaceutical orveterinary product, a medical device or a dietary product, that issuitable for use in accordance with the foregoing methods and uses ofthe second aspect of the present invention. The product comprises one ormore compounds having the structure of Formula A or B, or othercompounds of the invention as described further in section III.A of thisapplication below, optionally in combination with one or moreantibiotics or other antimicrobial agents as discussed herein in respectof the second aspect of the present invention. As discussed above, inthe option in which the product comprises one or more antibiotics orother antimicrobial agents, then they may be included in an amount,concentration and or with a release profile that is ordinarilysub-therapeutic or sub-optimally therapeutic for the treatment orprophylaxis of a microbial infection or colonization.

Also provided herein, is a method for the sensitization, and/or for thereduction in the tolerance, of one or more microorganisms to a selectedantimicrobial agent, the method comprising exposing the one or moremicroorganisms to one or more compounds having the structure of FormulaA or B, or other compounds of the invention as described further insection III.A of this application below. To put it another way, thesecond aspect of the present invention also provides for the use of oneor more compounds having the structure of Formula A or B, or othercompounds of the invention as described further in section III.A of thisapplication below to increase the sensitivity and/or reduce thetolerance, of one or more microorganisms to a selected antimicrobialagent. The microorganisms may, or may not, be microorganisms that areresistant to the selected antimicrobial agent. Thus, in one option, thein vivo and/or in vitro growth of the one or more microorganisms mayusually be unaffected by the selected antimicrobial agent (either atall, or at a selected concentration) in the absence of the one or morecompounds having the structure of Formula A or B, or other compounds ofthe invention as described further in section III.A of this applicationbelow, whereas exposure to the one or more compounds can cause the invivo and/or in vitro growth of the one or more microorganisms to bereduced (e.g. by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99% or substantially 100%) by exposure to the selectedantimicrobial agent (either at all, or at the selected concentration).In that context, a “selected concentration” includes concentrations thatare pharmaceutically and medically acceptable for use with patientsand/or animals, and lower concentrations such as 1%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% lower, whichmay or may not be sub-therapeutic. In another option, it may already bepossible to reduce the in vivo and/or in vitro growth of the one or moremicroorganisms by exposure to the antimicrobial agent (either at all, orat a selected concentration) in the absence of the one or more compoundshaving the structure of Formula A or B, or other compounds of theinvention as described further in section III.A of this applicationbelow, whereas exposure to the one or more compounds can cause anincrease in sensitivity and/or reduction in tolerance to theantimicrobial agent such that the in vivo and/or in vitro growth of theone or more microorganisms is further reduced (e.g. by 1%, 2%, 3%, 4%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% orsubstantially 100%) by the antimicrobial agent and/or an equivalentlevel of reduction of growth can be achieved with a lower concentrationor amount of the antimicrobial agent (e.g. using an amount (in weight)or concentration that is reduced by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or substantially 100%) and/or theperiod of treatment may be shortened, e.g. by 1, 2, 3, 4, 5, 6, 7 ormore days.

A further embodiment provides a method for the preparation of a productper se, such as a pharmaceutical or veterinary product, a medical deviceor a dietary product, that is suitable for use in accordance with theforegoing methods and uses of the second aspect of the presentinvention. The method may include the step of mixing the one or morecompounds having the structure of Formula A or B, or other compounds ofthe invention as described further in section III.A of this applicationbelow without one or more further components of the product, and therebyforming the product. The method may include forming the product(optionally without the one or more compounds), and then spraying orotherwise applying the one or more compounds having the structure ofFormula A or B, or other compounds of the invention as described furtherin section III.A of this application below, to the product. The methodmay include forming the product (optionally without the one or morecompounds), and then coating the product with the one or more compoundshaving the structure of Formula A or B, or other compounds of theinvention as described further in section III.A of this applicationbelow, for example, as described further below in the context ofcoatings.

The one or more compounds having the structure of Formula A or B, orother compounds as described further in section III.A of thisapplication below (“Component 1”) may be administered simultaneously,separately or sequentially with the one or more antibiotics and/or otheranti-microbial agents (“Component 2”).

In the context of simultaneous administration, Components 1 and 2 may bepresent in the same product for administration to the patient or animal.Alternatively, Components 1 and 2 may be present in separate productswhich are administered at the same time, although this may be via thesame of different routes. For example, both of Component 1 and 2 may beadministered, in separate products but at the same time, through anenteral route. In another embodiment, Component 1 may be administered byan enteral route, and Component 2 may be administered at the same timeby a parenteral route. In another embodiment, Component 1 may beadministered by a parenteral route, and Component 2 may be administeredat the same time by an enteral route. In another embodiment, both ofComponent 1 and 2 may be administered, in separate products but at thesame time, through a parenteral route.

In the context of separate and/or sequential administration, Components1 and 2 are administered to the patient at different times. Component 1may be administered before Component 2, or Component 2 may beadministered before Component 1. Preferably, the period of time betweenthe administration of Components 1 and 2 is less than the time taken bythe subject to clear an effective amount of the first-administeredcomponent, such that effective amounts of Components 1 and 2 will bepresent in the subject simultaneously. However, this may not beessential. In any case, the time taken by the subject to clear aneffective amount of the first-administered component will vary dependenton the nature of the component, the route of administration and theadministered form which may, for example, be a slow, delayed or extendedrelease formulation, product or device. The administration of Components1 and 2 (in either order) may be temporally separated by up to, about,or at least, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds,1 minute, 5 minutes 10 minutes, 20 minutes, 30 minutes 40 minutes 50minutes 1 hour, 2 hours, 3 hours, 4 hours 5 hours, 6 hours, 7 hours 8hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20hours 22 hours 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2weeks, 3 weeks, 1 month or more. Sequential administration includes themeaning of repeated and alternating administrations of Components 1 and2 (in either order), in which the administration of either or bothcomponents may be repeated any number of times, such as twice, threetimes, four times, five times, 10 times, 20 times, 30 times or more.

Repeated administration of either, or both components, whetheradministered simultaneously, separately or sequentially, may occur asoften as is therapeutically necessary, and can include continuousadministration (e.g. by intravenous infusion), of administration up to,about, or at least, every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 24 or 24 hours, every 1, 2, 3, 4, 5, 6, 7days, or every 1, 2, 3, 4 or more weeks, throughout the period oftreatment.

The period of treatment in accordance with the second aspect of thepresent invention is typically selected to achieve a therapeutically orprophylactically effective outcome, and will be judged accordingly, bythe skilled professional. Example of some suitable periods for treatmentcan include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, about1, 2, 3, or 4 weeks, or longer.

C. Inhibition of Formation, and Treatment of Preformed, Biofilms

A third aspect of the present invention is based on the surprisingfinding that compounds having the structure of Formula A or B, or othercompounds of the invention of the present invention as described furtherin section III.A of this application, have a broad range of action intreating and dispersing pre-existing biofilms, and inhibiting thedevelopment of biofilms, created by a wide range of bacterial and othermicrobial sources, and that this action is effective in a diverse arrayof environments.

Accordingly, a third aspect of the present invention provides a methodof inhibiting biofilm buildup, and/or disrupting a pre-existing biofilm,in or on a subject or article in need thereof the method comprisingadministering to the subject or article an effective amount of one ormore compounds having the structure of Formula A or B, or othercompounds of the invention of the present invention as described furtherin section III.A of this application.

Put another way, the third aspect of the present invention provides forthe use of one or more compounds having the structure of Formula A or B,or other compounds of the invention of the present invention asdescribed further in section III.A of this application for inhibitingbiofilm buildup, and/or disrupting a pre-existing biofilm, in or on asubject or article in need thereof.

In one embodiment, the one or more compounds having the structure ofFormula A or a salt and/or hydrate thereof, or a functional variantthereof, for use in accordance with the third aspect of the presentinvention are selected from the group consisting of a complex of anamino acid or an α-hydroxy acid with Fe III, such as a complex of quinicacid with Fe III, a complex of L-tyrosine with Fe III, a complex ofL-DOPA with Fe III, a complex of L-phenylalanine with Fe III, thecompounds represented by Formula I, Formula II, Formula III, Formula IV,Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X,Formula XI, Formula XII, Formula XIII, Formulas XIV, a compound selectedfrom the group consisting of a compound that binds to major outermembrane proteins (MOMPs) or FlaA of Campylobacter, a synthetic humanhisto-blood group antigen, a mimetic of human histo-blood group antigenor a synthetic sugar. Particularly preferred compounds may, or may not,include Fe-QA, Fe-Tyr, and/or Fe-DOPA.

1. Organisms to be Treated, Inhibited, or Killed

“Biofilm” as used herein refers any group of microorganisms in whichcells stick to each other on a surface.

Formation of a biofilm begins with the attachment of free-floatingmicroorganisms to a surface. These first colonists adhere to the surfaceinitially through weak, reversible adhesion via van der Waals forces. Ifthe colonists are not immediately separated from the surface, they cananchor themselves more permanently using cell adhesion structures suchas pill. Some species are not able to attach to a surface on their ownbut are sometimes able to anchor themselves to the matrix or directly toearlier colonists. It is during this colonization that the cells areable to communicate via quorum sensing. Once colonization has begun, thebiofilm grows through a combination of cell division and recruitment.Polysaccharide matrices typically enclose bacterial biofilms. The finalstage of biofilm formation is known as dispersion, and is the stage inwhich the biofilm is established and may only change in shape and size.

In one embodiment, a biofilm may comprise, consist essentially of, orconsist of, microbial cells growing in a biofilm that arephysiologically distinct from planktonic cells of the same organism,which, by contrast, are single-cells. Optionally, a biofilm maycomprise, consist essentially of, or consist of, one species or strainof bacterial cell.

In an alternative option, a biofilm may comprise, consist essentiallyof, or consist of, more than one species or strains of bacterial cell,such as up to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,60 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000 or more different species or strains of bacterial cell.

The bacterial species or strains in biofilms can include bacteriaselected from one or more of gram negative, gram positive, aerobic andanaerobic bacteria and/or archaea.

Accordingly, compositions and methods for inhibiting, reducing, orremoving biofilm forming bacteria and bacterial infections are providedby the third aspect of the present invention.

In accordance with some embodiments of the third aspect of the presentinvention, the biofilm forming bacteria to be inhibited, reduced,removed, or treated may be gram-negative and/or gram-positive bacteria,such as Pseudomonas aeruginosa, Campylobacter jejuni, Helicobacterpylori, Escherichia coli, Enteropathogenic Escherichia coli (EPEC),Uropathogenic Escherichia coli (UPEC), Staphylococcus epidermidis,Staphylococcus aureus, and Enterococcus faecalis.

The following are representative organisms that can be killed or growthinhibited, or their ability to produce or maintain biofilms degraded,reduced, inhibited or prevented in accordance with the third aspect ofthe present invention.

One form of biofilm of particular interest in certain embodiments of thethird aspect of the present invention is biofilm that forms dentalplaque. The effectiveness of the present invention against dental plaqueis demonstrated in Example 17. The biofilm in dental plaque typicallycomprises a variety of microbial organisms, including both aerobic andanaerobic bacteria, and typically includes over 700 different species ofbacteria and archaea. Dental plaque biofilms are responsible for many ofthe diseases common to the oral cavity including dental caries,periodontitis, gingivitis, and the less common peri-implantitis (similarto periodontitis, but with dental implants), however biofilms can bepresent on healthy teeth as well.

Accordingly, the third aspect of the present invention also providesmethods and uses for preventing or inhibiting the formation of, fortreating, or for reversing or removing conditions including dentalplaque, dental caries, periodontitis, gingivitis, and the less commonperi-implantitis. Said method or use may comprise administering acomposition according to the third aspect of the present invention tothe mouth of a subject, thereby to achieve the intended effect.

In the context of dental products, and in the context of inhibitingand/or removing dental plaques, an effective concentration of 340 μM isdemonstrated in Example 17, although higher or lower concentrations ofthe one or more compounds according to section III.A below may also besuitable. For example, dental products may present the buccal cavity orteeth with one or more of said compounds at a concentration within therange of about 1 μM to about 1M, such as about, or up to, 10 μM, 20 μM,30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM,130 μM, 140 μM, 15 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 210 μM,220 μM, 230 μM, 240 μM, 250 μM, 260 μM, 270 μM, 280 μM, 290 μM, 300 μM,310 μM, 320 μM, 330 μM, 340 μM, 350 μM, 360 μM, 370 μM, 380 μM, 390 μM,400 μM, 410 μM, 420 μM, 430 μM, 440 μM, 450 μM, 460 μM, 470 μM, 480 μM,490 μM, 500 μM, 510 μM, 520 μM, 530 μM, 540 μM, 550 μM, 560 μM, 570 μM,580 μM, 590 μM, 600 μM, 610 μM, 620 μM, 630 μM, 640 μM, 650 μM, 660 μM,670 μM, 680 μM, 690 μM, 700 μM, 710 μM, 720 μM, 730 μM, 740 μM, 750 μM,760 μM, 770 μM, 780 μM, 790 μM, 800 μM, 810 μM, 820 μM, 830 μM, 840 μM,850 μM, 860 μM, 870 μM, 880 μM, 890 μM, 900 μM, 910 μM, 920 μM, 930 μM,940 μM, 950 μM, 960 μM, 970 JAM, 980 μM, 99 μM, 1 mM, 2 mM, 3 mM, 4 mM,5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM 25 mM, 30 mM, 35 mM,40 mM, 45 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM,250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 600 mM, 700 mM, 800 mM,900 mM, 1M or more. Optionally, the concentration may be:

(a) up to 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30μM;

(b) within a range selected from the group consisting of from 35 to 335μM, 40 to 300 μM, 50 to 300 μM, 50 to 250 μM, 50 to 200 μM, 60 to 300μM, 60 to 250 μM, 60 to 200 μM, 80 to 300 μM, 80 to 250 μM, 80 to 200μM, 100 to 300 μM, 100 to 250 μM, or 100 to 200 μM; or

(c) at least, or about, 345 μM, 350 μM, 360 μM, 370 μM, 380 μM, 390 μM,400 μM, 450 μM, 0.5 mM, 1 mM, 2 mM or more.

Optionally the concentration of the one or more compounds may be withina range selected from the group consisting of from about 1 μM to about 1mM, or about 30 μM to about 0.5 mM, or about 60 μM to about 0.4 mM.

Another form of biofilm of particular interest to the third aspect ofthe present invention is biofilm on medical devices, including contactlenses. Biofilms on contact lenses may, for example, comprise, consistessentially of; or consist of one or more bacteria selected fromArchromobacter, Delftia, Staphylococcus, Stenotrophomonas, andStreptococci species, and Pseudomonas aeruginosa.

Another form of biofilm of particular interest in the present inventionis biofilms formed on the skin, for example biofilms which comprise,consist essentially of, or consist of Propionibacterium acnes.Accordingly. the third aspect of the present invention also providesmethods and uses for preventing or inhibiting the formation of, fortreating, or for reversing or removing acne and othermicrobially-induced skin conditions, including recalcitrant and/oranti-biotic resistant conditions, the method or use comprising thetopical administration of a composition according to the third aspect ofthe present invention to the skin of a subject, thereby to achieve theintended effect.

Another form of biofilm which may, or may not, be of particular interestin the third aspect of the present invention is biofilms that comprise,consist essentially of, or consist of, epsilon proteobacteria class,such as the spirilloid Wolinella spp., Helicobacter spp., and mostparticularly Campylobacter spp. In one embodiment, the application ofthe third aspect of the present invention to biofilms that comprise,consist essentially of, or consist of, Campylobacter spp. may, or inanother option may not, be of interest. Many other types of biofilms areof interest for the present invention, further examples of which arediscussed in further sections of this application.

Campylobacter are gram negative, spiral rod shaped bacteria with asingle flagellum at one or both poles. They belong to the epsilonproteobacteria class and are closely related to Helicobacter andWolinella. At least a dozen species of Campylobacter have beenimplicated in human disease, with C. jejuni and C. coli the most common.

Campylobacter jejuni is the major cause of human bacterialgastroenteritis (Pearson, et al., Appl Environ Microbiol., 59:987-996(1993)). The four major sources of infection are raw meat (particularlypoultry), untreated water, raw milk, and pets (Humphrey, et al., J ApplBacteriol. 61:125-132. (1986) and Skirrow, Int J Food Microbiol.,12:9-16 (1991)). It has also been suggested that, although notuniversally the case (Humphrey, et al., Public Health Lab Serv MicrobiolDigest., 13:86-88. 91996), Jacobs-Reitsma, et al., Epidemiol Infect.,114:413-421 (1995), and Lindblom, et al., J Hyg., 96:385-391 (1986)),survival in the water systems of animal husbandry facilities andanimal-processing units promotes infection in animals andcross-contamination of animal carcasses (Humphrey, et al., EpidemiolInfect., 98:263-269 (1987), Kazwala, et al., Vet Rec. 1990; 126:305-306.(1990) and, Pearson, et al., Appl Environ Microbiol., 59:987-996(1993)). Thus, the survival of C. jejuni in aquatic environments isimportant both directly and indirectly in the causation of humandisease.

Campylobacter spp. have outer membrane proteins (OMPs) (Buchanan, Curr.Opin. Struc. Biol., 9(40:455-461 (1999); Huyer, et al., FEMS Microbiol.Lett., 37(3):247-250 (1986)]. The major outer membrane proteins (MOMPs)have unique structural features, and function as porins which arehelpful for linking up the bacteria and their environment. Campylobacterspp. possess polar flagella which provide the necessary motility forintestinal colonization. The flagellin gene of Campylobacter has twosimilar copies: flaA and flaB. The length of coding regions for the flaAand flaB sequences are both around 1.7 kilobases, and flaA and flaBsequences locate about 180 bases apart from each other (Meinersmann, etal., Microbiology, 146(9):2283 (2000)).

In one embodiment of the third aspect of the present invention, thedisclosed compositions bind to major outer membrane proteins (MOMPs) orFlaA of Campylobacter and prevent the bound MOMPs and bound FlaA frombinding or associating with their ligands on: other Campylobacterbacteria; other species of bacteria; biofilm or biofilm components; orto surfaces. By binding to the MOMPs and FlaA, the compounds inhibit thebacteria from binding to surfaces or each other to produce biofilm. Theinhibition of binding can be accomplished by interfering with thebinding of natural ligands of MOMPs or FlaA or by physically inhibitingthe association of the bacteria expressing MOMPs or FlaA to otherorganisms or surfaces.

In another embodiment of the third aspect of the present invention, thedisclosed compositions also bind to the MOMP protein of Campylobacterwhen MOMP has been mutated to prevent O-glycosylation by mutation ofThr-268 to glycine to form MOMP-T (also referred to as MOMP^(T268G)). Asshown in Table 1, expression of the MOMP^(T268G) protein has been foundto increase 10-fold compared with wildtype. Treatment of theMOMP^(T268G) strain with the compositions does not impact planktonicgrowth, but does partially inhibit biofilm formation demonstrating thecompositions bind to the non-glycosylated MOMP with lower affinity.

As shown in Table 1, expression of the MOMP^(T268G) protein has beenfound to increase 10-fold compared with wildtype. Regardless of whetherMOMP is glycosylated or not, the compositions disclosed herein are stilleffective against mixed populations of glycosylated and non-glycosylatedCampylobacter. In a mixed population of glycosylated andnon-glycosylated forms, the wildtype glycosylated form of Campylobactergreatly outcompetes the mutant non-glycosylated form, and over time thenon-glycosylated bacteria disappear and the glycosylated bacteria becomethe only bacteria present.

Biofilms are usually found on solid substrates submerged in or exposedto an aqueous solution, although they can form as floating mats onliquid surfaces. Biofilms can form on a myriad of surfaces. For example,biofilms can grow in showers very easily since they provide a moist andwarm environment for the biofilm to thrive. Biofilms can form insidewater and sewage pipes and cause clogging and corrosion. Biofilms onfloors and counters can make sanitation difficult in food preparationareas. Biofilms can form in cooling- or heating-water systems and areknown to reduce heat transfer in these systems

One method, or use, in accordance with a third aspect of the presentinvention includes administering an effective amount of the one or morecompounds as defined in section III.A of this application to a subjectin need thereof, to inhibit biofilm formations, or alternatively, toreduce and/or remove biofilm formation. The one or more compounds may beadministered alone, or in combination with an antimicrobial agent, suchas an antibiotic.

In certain embodiments of the third aspect of the present invention, inthe context of the treatment of subjects (such as humans or animals) itmay be desirable to provide continuous delivery of one or more compoundsto a subject in need thereof. For intravenous or intra-arterial routes,this can be accomplished using drip systems, such as by intravenousadministration. For topical applications, repeated application can bedone or a patch can be used to provide continuous administration of thecompounds over an extended period of time. For example, the compoundsmay be delivered to a chronic wound from a wound dressing. The dressingmay also contain one or more antibiotics, and if necessary the wounddressing may be changed frequently. The compounds may also be deliveredin a conjugated form (for example, as shown in FIGS. 15A-C and FIGS. 16Aand B) so that they are immobilized on a surface.

In other embodiments of the third aspect of the present invention, themethod includes contacting a surface with an effective amount of thecompounds, to inhibit biofilm buildup, reduce built up biofilm, and/orremove built up biofilm. “Contacting” includes, but is not limited to,touching, impregnating, compounding, mixing, integrating, coating,spraying, dipping, flushing, irrigating, and wiping. In certainembodiments, it may be desirable to provide continuous delivery of oneor more compounds to the surface or system being treated. Thecompositions can be used to coat, impregnate, flush, or rinse a surfaceof tubing or a medical device, especially an insertable medical device.Tubing includes, but is not limited to, disposable, permanent, andindwelling catheters, long term urinary devices, tissue bonding urinarydevices, wound drain tubes, ventricular catheters, endotracheal tubes,breathing tubes, feeding tubes, dairy lines, oil and gas pipeline anddrinking water lines. When an object is tubing (e.g., dental unitwaterline, a dairy line, a food and beverage processing line, etc.), acomposition may be poured into the tubing and both ends of the tubingclamped such that the composition is retained within the lumen of thetubing. The tubing is then allowed to remain filled with the compositionfor a period of time sufficient to remove substantially all of themicroorganisms from at least one surface of the object, generally, forat least about 1 minute to about 48 hours. Alternatively, tubing may beflushed by pouring a composition into the lumen of the tubing for anamount of time sufficient to prevent substantial growth of all biofilmembedded microorganisms. Such flushing may be required only once, or maybe required at regular intervals over the lifetime of use of the tubing.Concentrations of active components in a composition may vary as desiredor necessary to decrease the amount of time the composition is incontact with a medical device.

The methods allow disinfection, inhibition, or prevention of biofilmformation on the surfaces being treated and reduction of transmission ofbiofilm forming microorganisms from the surface to another surface. Thenumber of the bacterial colony forming units (cfu) on the surface beingtreated with the compounds may be reduced by 50%, by 60%, by 70%, by80%, by 90% or by 100%, or, the buildup of bacterial colony formingunits on the treated surface may be reduced by 50%, by 60%, by 70%, by80%, by 90% or by 100%.

In one embodiment of the third aspect of the present invention,compositions and articles, including but not limited to pharmaceuticaland veterinary compositions, food or feed additive compositions, anddental products including chews may be prepared from the one or morecompounds as defined above, optionally formulated and/or used incombination with one or more antibiotics or other anti-microbial agents,and these compositions may further be used for the treatment orprophylaxis of a microbial infection or biofilm formed by bacteria orother microorganisms, including one or more of the following: S.epidermidis, E. faecalis, E. coli, S. aureus includingVancomycin-resistant Staphylococcus aureus (VRSA) andMethicillin-resistant Staphylococcus aureus (MRSA), EnteropathogenicEscherichia coli (EPEC), Uropathogenic Escherichia coli (UPEC),Pseudomonas, Streptococcus pneumoniae, Streptococcus anginosus,Neisseria gonorrhoeae, Salmonella (including drug-resistantnon-typhoidal, Salmonella including drug-resistant Salmonella serotypetyphi, Salmonella Enteritidis, Salmonella Typhimurium, Mycoplasma,Eimeria, Enterococci, Shigella, Vancomycin-resistant Enterococcus (VRE),Erythromycin-resistant Group A Streptococcus, Clindamycin-resistantGroup B Streptococcus, Carbapenem-resistant Enterobacteriaceae (CRE),drug-resistant tuberculosis, Extended spectrum Enterobacteriaceae(ESBL), multidrug-resistant Acinetobacter (including MRAB), Clostridiumdifficile, Enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa,Brachyspira, Propionibacterium acnes, and Clostridium perfringen.

2. Methods of Administration

In one embodiment, the compounds and formulations, derivatives thereofand combinations thereof for use in accordance with the third aspect ofthe present invention can be administered topically to a subject in needthereof in an effective amount to prevent or treat a microbialinfection, by inhibiting buildup of biofilm or to reduce and/or removebuilt up biofilm.

Any suitable topical formulation can be used, for example as describedin Section III.C.3 of this application, below, including emulsions (suchas those described in section III.C.3(a)), lotions (such as thosedescribed in section III.C.3(b)), creams (such as those as described insection III.C.3(c)), ointments (such as those described in sectionIII.C.3(d)), gels (such as those described in section III.C.3(e)), orfoams (such as those described in section III.C.3(f)).

The compositions may be used alone or in combination with knownantimicrobial agents, such as those described further below in sectionIII.B of this application. As such, compositions described in respect ofthe second aspect of the present invention may also be useful in thepractice of the third aspect of the present invention.

The compositions are useful for treating topical conditions caused bybiofilm buildup by microorganisms including, but not limited togram-negative and gram-positive bacteria, including Staphylococcus(including, but not limited to S. aureus and Staphylococcusepidermidis), Pseudomonas, E. coli., Streptococcus pyogenes (Reviewed inNusbaum, et al., Skin Therapy Lett., 17(7):1-5 (2012)),Propionibacterium acnes and Streptococcus anginosus.

In some embodiments the compositions are used as a topical antibacterialmedication for skin infections caused by methicillin-resistantStaphylococcus aureus. Methicillin-resistant Staphylococcus aureus(MRSA) is a bacterium that is resistant to many antibiotics. Thespectrum of disease caused by MRSA appears to be similar to that ofStaphylococcus aureus in the community. Soft tissue infections (SSTIs),specifically furuncles (abscessed hair follicles or “boils”), carbuncles(coalesced masses of furuncles), and abscesses, are the most frequentlyreported clinical manifestations.

The most common manifestations of community associated-MRSA are simpleskin infections, such as impetigo, boils, abscesses, folliculitis, andcellulitis. Others include children with minor skin infections (such asimpetigo) and secondarily infected skin lesions (such as eczema, ulcers,or lacerations). The compositions can also be used to treat MRSAinfections of the CNS, which include, but are not limited to Meningitis,Brain abscess, subdural empyema, spinal epidural abscess. Reviewed inLiu, et al., Clin Infect Dis., 52(3):e18-55 (2011).

Additional examples of conditions that can be treated include atopicdermatitis, acne, bullous and non-bullous impetigo, pemphigus foliaceus,miliaria, feruncles (also known as boils) and chronic wounds such asdiabetic foot ulcers, venous insufficiency ulcers, and pressure ulcers.

In the context of treating acne, an effective concentration of 34 μM isdemonstrated in Example 24, although higher or lower concentrations ofthe one or more compounds according to section III.A below may also besuitable for the treatment of acne and any of the other skin conditionsas discussed herein. For example, the treatment of these skin conditionsin accordance with the present invention may utilize one or more of saidcompounds at a concentration within the range of about 1 μM to about 1M,such as about, or up to, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 15 μM, 160 μM,170 μM, 180 μM, 190 μM, 200 μM, 210 μM, 220 μM, 230 μM, 240 μM, 250 μM,260 μM, 270 μM, 280 μM, 290 μM, 300 μM, 310 μM, 320 μM, 330 μM, 340 μM,350 μM, 36 μM, 370 μM, 380 μM, 390 μM, 400 μM, 410 μM, 420 μM, 430 μM,440 μM, 450 μM, 460 μM, 470 μM, 480 μM, 490 μM, 500 μM, 510 μM, 520 μM,530 μM, 54 μM, 550 μM, 560 μM, 570 μM, 580 μM, 590 μM, 600 μM, 610 μM,620 μM, 630 μM, 640 μM, 650 μM, 660 μM, 670 μM, 680 μM, 690 μM, 700 μM,710 μM, 720 μM, 730 μM, 740 μM, 750 μM, 760 μM, 770 μM, 780 μM, 790 μM,800 μM, 810 μM, 820 μM, 830 μM, 840 μM, 85 μM, 860 μM, 870 μM, 880 μM,890 μM, 900 μM, 910 μM, 920 μM, 930 μM, 940 μM, 950 μM, 960 μM, 970 μM,980 μM, 990 μM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10mM, 15 mM, 20 mM 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70 mM,80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM,450 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1M or more. Optionally,the concentration may be:

(a) up to 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30μM;

(b) within a range selected from the group consisting of from 35 to 335μM, 40 to 300 μM, 50 to 300 μM, 50 to 250 μM, 50 to 200 μM, 60 to 300μM, 60 to 250 μM, 60 to 200 μM, 80 to 300 μM, 80 to 250 μM, 80 to 200μM, 100 to 300 μM, 100 to 250 μM, or 100 to 200 μM; or

(c) at least, or about, 345 μM, 350 μM, 360 μM, 370 μM, 380 μM, 390 μM,400 μM, 450 μM, 0.5 mM, 1 mM, 2 mM or more.

Optionally the concentration of the one or more compounds may be inwithin a range selected from the group consisting of from about 1 μM toabout 1 mM, or about 30 μM to about 0.5 mM, or about 60 μM to about 0.4mM.

Atopic dermatitis (AD) affects 10-20% of children with 60% of casesoccurring within a child's first year and 85% before the age of 5(Krakowski, et al., Pediatrics, 122(4):812-24 (2008)). Many casespersist into adulthood as evidenced by the 1-3% prevalence of AD amongthe adult population (Leung, et al., Lancet, 361(9352):151-60 (2003)).AD patients are colonized with S. aureus and this organism has beenshown to exist in both dry skin as well as areas of severe dermatitis(Ikezawa, et al., Allergy Asthma Immunol Res., 2(4):235-46 (2010)).Disease severity has been directly correlated to the degree of S. aureuscolonization and therapy generally fails to improve symptoms in thepresence of high S. aureus counts (Akiyama, et al., J Dermatol Sci.,23(3):155-6 (2000)). Confocal laser scanning micro has demonstrated thepresence of biofilms in skin stripping and biopsy specimens from ADpatients (Akiyama, et al., Br J Dermatol., 148(3):526-32 (2003)). Thepresence of S. aureus biofilms have been shown in specimens of bullousimpetigo and pemphigus foliaceus (Akiyama, et al., Br J Dermatol.,148(3):526-32 (2003)) while biofilms containing both S. aureus andStreptococcus pyogenes have been identified in non-bullous impetigo(Akiyama, et al., J Dermatol Sci., 32(3):193-9 (2003)). The difficultyin eradicating S. aureus colonization with conventional antibiotictherapy may be due to the presence of biofilms. Biofilm formation hasalso been demonstrated in a murine model inoculated with S. aureusisolated from a furuncle (Yamasaki, et al., J Antimicrob Chemother.,48(4):573-7 (2001)).

Biofilms have been implicated in miliaria by a clinical study in whichonly extracellular polymeric substance (EPS) producing S. epidermidiswas capable of inducing lesions after inoculation and occlusion (Mowad,et al., J Am Acad Dermatol., 33(5 Pt 1):729-33 (1995)). Biopsy specimensrevealed sweat glands blocked with EPS material, further supporting apathogenic role for biofilms in this condition. Several factors, forexample, firm adherence of dermatophytes to the nail plate, presence ofdormant fungal elements, ability of yeast to form biofilms, anddifficulty of eradication all suggest that biofilm involvement inonychomycosis (Burkhart, et al., J Am Acad Dermatol., 47(4):629-31(2002)).

Chronic wounds present an optimal environment for microbialproliferation. In a clinical study of 66 wounds of various etiologies,60% of chronic wounds were shown to contain biofilms as compared to 6%of acute wounds, indicating a role of biofilms in wound chronicity.Traditional cultures identified Staphylococcus, Pseudomonas, andEnterococcus as the predominant organisms (James, et al., Wound RepairRegen., 16(1):37-44 (2008).

In a preferred embodiment, the compounds may be incorporated into woundirrigation solutions. In another preferred embodiment, the compounds maybe incorporated into cosmetic formulations.

The compositions of the compounds according to the third aspect of thepresent invention are also useful in oral health for both prophylaxisand treatment of infections. For example, the compounds may be used totreat or prevent infections in dental pulp by Streptococcus anginosus,or prevent attachment of biofilms to tooth surfaces. The compounds maybe applied directly to tooth surfaces or applied to dental pulp during aprocedure. The compounds may also be incorporated into dental productssuch as toothpaste, mouthwash, floss, toothpicks, and chewable products(including food products), a mouth shield, a dental instrument,dentures, dental retainers, dental braces including plastic braces (suchas Invisalign), bristles of toothbrushes, dental prostheses andorthodontic devices, chewable non-food items, or foods, as well asapplied as coatings directly to dental tissues. The compositions may beused for dental care of both humans and animals, including pets such asdogs and cats as well as livestock and horses. For example, thecompounds may be incorporated into chewable foods or toys, such as dogbones and biscuits.

In fact, in one embodiment of particular interest to the presentinvention, there is provided a human or animal (especially a dog) chewcomposition comprising one or more compounds as defined in SectionIII.A. Exemplary dog and other animal chews which can be modified toinclude the one or more compounds as defined in Section III.A includethose described in U.S. Pat. No. 6,086,940, the contents of which areincorporated herein by reference. Further exemplary chews include theOravet® dental hygiene chew produced by Merial (seehttp://merial.com/en/press-releases/merial-introduces-oravet-dental-hygiene-chews-for-dogs/,the contents of which are incorporated herein by reference) and theKaNoodles dental chews (see http://kanoodlesusa.com/, the contents ofwhich are incorporated herein by reference). Dental chews in accordancewith the present invention can be used in dogs and other animals toinhibit the production of biofilms that form plaque, and/or to reduce ortreat or prophylactically treat halitosis. Chewing said chews may alsohelp scrub away existing plaque and/or calculus. Optionally, the chewsmay be usefully used regularly, such as daily and optionally daily afterone or more meals.

The compounds may, in accordance with the third aspect of the presentinvention, be added to drinking water or other drinkable fluids.

Other modes of administration in accordance with the third aspect of thepresent invention can include:

(i) Parenteral administration, which may include administration to apatient intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intravitreally,intratumorally, intramuscularly, subcutaneously, subconjunctivally,intravesicularly, intrapericardially, intraumbilically, by injection,and by infusion, for example as further described in Section III.C.1 ofthis application, below. Parenteral administration can include the useof formulations as described herein which are formulated for controlledrelease including immediate release, delayed release, extended release,pulsatile release, and combinations thereof, as further described inSection III.C.1(a) of this application, below.

(ii) The compounds can be incorporated into injectable/implantable solidor semi-solid implants, such as polymeric implants, for example asfurther described in Section III.C.1(b) of this application, below.

(iii) Enteral administration, including administration in the form ofsuitable oral dosage forms such as tablets, capsules, solutions,suspensions, syrups, and lozenges, for example, as further described inSection III.C.2 of this application, below. Optionally, enteraladministration may include administration of controlled release enteralformulations, including oral dosage forms, such as capsules, tablets,solutions, and suspensions, which are formulated for controlled release,including extended and/or delayed release, such as described in moredetail below in Section III.C.2(a) of this application.

(iv) The administration of one or more disinfecting formulations orcleaning formulations, such as those described in Section III.C.4 ofthis application, below.

3. Hospital and Other Environments

The methods and uses of the third aspect of the present invention may bepracticed in the hospital and also in other medical and non-medicalenvironments in order to address, inhibit, treat, ameliorate and/ordisrupt biofilms. Further examples of microbial infection andcolonizations and biofilm formations that can be addressed by the thirdaspect of the invention are discussed further below, and also furtherdefine medical uses and methods in accordance with the third aspect ofthe present invention for the treatment and/or prophylaxis of subjects(including humans and animals) in need thereof.

For example, S. epidermidis contributes to biofilms that grow on plasticdevices placed within the body (Otto, Nature Reviews Microbiology,7(8):555-567 (2009)). This occurs most commonly on intravenous cathetersand on medical prostheses (Hedin, Scandinavian Journal of InfectiousDiseases Supplementum, 90:1-59 (1993)). Infection can also occur indialysis patients or anyone with an implanted plastic device that mayhave been contaminated. Another disease it causes is endocarditis. Thisoccurs most often in patients with defective heart valves. In some othercases, sepsis can occur in hospital patients.

As a further example, Methicillin-resistant S. aureus (MRSA), is one ofa number of greatly feared strains of S. aureus which have becomeresistant to most β-lactam antibiotics. MRSA strains are most oftenfound associated with institutions such as hospitals, but are becomingincreasingly prevalent in community-acquired infections. A recent studyby the Translational Genomics Research Institute showed that nearly half(47%) of the meat and poultry in U.S. grocery stores were contaminatedwith S. aureus, with more than half (52%) of those bacteria resistant toantibiotics (ScienceDaily, 15 Apr. 2011).

In another example, Enterococcus faecalis causes many of the antibioticresistant infections in hospitals, a consequence of its inherentresistance to certain antibiotics and its ability to survive andproliferate in the intestinal tract. Escherichia coli is one of the mostfrequent causes of many common bacterial infections, includingcholecystitis, bacteremia, cholangitis, urinary tract infections otherclinical infections such as neonatal meningitis and pneumonia. Forexample, the compositions can be used to treat (for example, as adjuncttherapy) conditions caused by community- and/or hospital-acquiredurinary tract infections (UTI's) caused by strains of Escherichia coli(drug resistant or otherwise) in immunocompromised patients.

In accordance with a further example, the aggressive colonization ofstainless steel surfaces by P. aeruginosa for example, apart from beingof enormous industrial significance, is also of medical relevance; P.aeruginosa infections are prevalent in burn units where large stainlesssteel tubs, known as hydrotherapy units, are often used to treatpatients with severe burns.

Antibiotics are largely ineffective in clearing biofilms, although thethird and second aspects of the present invention may be combined inorder to potentiate the effect of antibiotics.

The most common treatment for these infections is to remove or replacethe infected implant, though in all cases, prevention is ideal. The drugof choice is often vancomycin, to which rifampin or aminoglycoside canbe added. Hand washing has been shown to reduce the spread of infection.Accordingly, compositions in accordance with the third aspect of thepresent invention may include hand wash and/or hand spray compositions,and may be used accordingly in the treatment of hands and other bodysurfaces.

Preliminary research also indicates S. epidermidis is universally foundinside affected acne vulgaris pores, where Propionibacterium acnes isnormally the sole resident (Bek-Thomson, et al., J. Clin. Microbiol.,46(10):3355-3360 (2008).

a. Use as is Infection Agent

The one or more compounds for use in the third aspect of the presentinvention can, in accordance with a further embodiment, be used asdisinfection (or pesticide) agents (the United States EnvironmentalProtection Agency, “EPA”, defines biofilms as pestilent), for example,in high risk environments such as in hardware from hospitals orhealthcare facilities. As such, the one or more compounds may beformulated as a disinfecting formulation or cleaning formulation, suchas those described in Section III.C.4 of this application, below.

In accordance with a further embodiment of the third aspect of thepresent invention, there is provided a method or use comprising the useof the disinfection agent in high-risk environments such as in hardwarefrom hospitals or healthcare facilities, cosmetic, consumer andindustrial applications, to prevent biofilm buildup or reduce biofilmfrom a surface of interest. In these embodiments, the compounds may, forexample, be sprayed onto the surface in the form of a foam, solution orgel, or applied to the surface (wipe down) by means of a carrier forexample tissue, material or other porous item containing the one or morecompounds. A further embodiment of the third aspect of the presentinvention is a disinfection agent as described herein and also providedis a product or article treated with a disinfection agent as describedherein.

The World Health Organization (WHO) estimates that at any time, morethan 1.4 million people worldwide are affected by infections acquired inhospitals. Cleaning, disinfection and sterilization saves lives andimproves patient outcomes. Between 5% and 10% of patients admitted tomodern hospitals in the developed world acquire one or morehealthcare-associated infections. The Centers for Disease Control andPrevention (CDC) estimate that approximately 1.7 millionhealthcare-associated infections occur annually in hospitals in theUnited States, and are associated with nearly 100,000 deaths each year.Healthcare-associated infections are also an important problem inextended care facilities, including nursing homes and rehabilitationunits. Transmission of healthcare-associated pathogens most frequentlyoccurs via the hands of healthcare workers, who inadvertentlycontaminate their hands during various patient care activities. Lessfrequently, contaminated surfaces in healthcare facilities maycontribute to the spread of healthcare-associated pathogens.

The varying levels of disinfection used in a healthcare facility may bedefined by Spaulding's Classification (Sehulster, et al., Guidelines forenvironmental infection control in health-care facilities.Recommendations from CDC and the Healthcare Infection Control PracticesAdvisory Committee (HICPAC). Chicago Ill.; American Society forHealthcare Engineering/American Hospital Association; 2004.).Spaulding's levels, non-critical, semi-critical, and critical, are basedon the potential for infectious disease spread via equipment,instruments, and furniture as well as the level of sterility normallyrequired for the body part coming in contact with it. Levels ofdisinfection that correlate with Spaulding's classification are low,intermediate, high, and sterilization. The US Centers for DiseaseControl (CDC) has further delineated disinfection levels forenvironmental surfaces in its “Guidelines for Environmental InfectionControl in Health-Care Facilities”.

Critical items confer a high risk for infection if they are contaminatedwith any microorganism. Thus, the third aspect of the present inventionalso provides objects treated for sterilization as described herein,which objects enter sterile tissue or the vascular system and must besterile because any microbial contamination could transmit disease. Thiscategory includes surgical instruments, cardiac and urinary catheters,implants, and ultrasound probes used in sterile body cavities. Semicritical items contact mucous membranes or nonintact skin. This categoryincludes respiratory therapy and anesthesia equipment, some endoscopes,laryngoscope blades, esophageal manometry probes, cystoscopes, anorectalmanometry catheters, and diaphragm fitting rings. These medical devicesshould be free from all microorganisms; however, small numbers ofbacterial spores are permissible. Specific examples of critical or semicritical instruments include invasive endoscopes such as laparoscopes,and rigid instruments with no operating channel. Arthroscopes andlaparoscopes which are inserted into sterile body cavities as well asaccessory instrumentation should be sterile. Other examples includegastroscopes, duodenoscopes, sigmoidoscopes, proctoscopes, colonoscopes,bronchoscopes, and laryngoscopes.

The compounds may also be used in accordance with the third aspect ofthe present invention as food processing aids. For example, solutions ofthe one or more compounds as defined in section III.A below could besprayed on animal carcasses or products (include meat part products)derived therefrom (i.e. poultry, fish, and meat or others, for example,as described in respect of the first aspect of the present invention) toprevent or inhibit colonization by bacteria, or inactivate biofilmformation. The compounds could, for example, be applied by dippingchicken (or other animal) carcasses or product derived therefrom in acontainer of a solution of the compounds, or by spraying an animalcarcass with a solution of the compounds.

In a preferred embodiment, aqueous solutions of FeQ, FeTyr, FeDOPAand/or Fe-Phe may be used as food processing aids. After treatment, thecompounds may, if desired, be removed by washing.

A further embodiment of the third aspect of the present inventionprovides an animal carcass (such as a chicken or other poultry, fish orother meat) and/or products (include meat part products) derivedtherefrom which have been treated, for example by spraying or dipping,in accordance with the third aspect of the present invention, andoptionally wherein the one or more compounds are subsequently removedfully or partially by washing.

b. Use as a Coating

In other embodiments of the third aspect of the present invention, theone or more compounds having the structure of Formula A or B, or othercompounds of the invention of the present invention as described furtherin section III.A of this application, can be incorporated into coatingsused to coat medical devices, and other articles.

Accordingly, the third aspect of the present invention also provides amethod of coating a device or other article, comprising applying acoating comprising, consisting essentially of, or consisting of, one ormore compounds having the structure of Formula A or B, or othercompounds of the invention of the present invention as described furtherin section III.A of this application.

The third aspect of the present invention also provides coated devicesor articles, having a coating comprising, consisting essentially of, orconsisting of, one or more compounds having the structure of Formula Aor B, or other compounds of the invention of the present invention asdescribed further in section III.A of this application.

Suitable coating methods are known in the art. Methods for coatingmedical devices are disclosed for example in U.S. Publication Nos.20030054090 and 20120276280 and U.S. Pat. Nos. 5,879,697, 7,247,338 and8,028,646. The compounds can be applied to medical devices and otherarticles in any number of ways, including, but not limited to, ionicbinding to a surface coating, passive adsorption, or dispersion within apolymeric base material making up the surface of the device or coated onthe device surfaces (for example by dip coating, spray coating,ultrasonic spray coating, melt processing, application of films, solventcoating, etc.).

In a preferred embodiment, the one or more compounds are combined withpolymers, and coated on medical devices or other articles. Suitablepolymers include, but are not limited, to poly(lactides);poly(glycolides); poly(lactide-co-glycolides); poly(lactic acid);poly(glycolic acid); poly(lactic acid-co-glycolic acids);polycaprolactones; poly(orthoesters); polyanhydrides;poly(phosphazenes); polyhydroxyalkanoates [includingpoly-3-hydroxybutyrate, poly-3-hydroxybutyrate-co-3-hydroxyvalerate(PHBV), poly-4-hydroxybutyrate,poly-3-hydroxybutyrate-co-4-hydroxybutyrate]; synthetically orbiologically prepared polyesters (including polyesters with one or moreof the following monomeric units: glycolic, lactic; trimethylenecarbonate, p-dioxanone, or ϵ-caprolactone);poly(lactide-co-caprolactones); polyesters; polycarbonates; tyrosinepolycarbonates; polyamides (including synthetic and natural polyamides,polypeptides, and poly(amino acids)); polyesteramides; poly(dioxanones);poly(alkylene alkylates); polyethers (such as polyethylene glycol, PEG,and polyethylene oxide, PEO); polyvinyl pyrrolidones or PVP;polyurethanes; polyetheresters; polyacetals; polycyanoacrylates;poly(oxyethylene)/poly(oxypropylene) copolymers; polyacetals,polyketals; polyphosphates; (phosphorous-containing) polymers;polyphosphoesters; polyalkylene oxalates; polyalkylene succinates;poly(maleic acids); chitin; chitosan; modified chitosan; collagen; silk;biocompatible polysaccharides; biocompatible copolymers (including blockcopolymers or random copolymers); hydrophilic or water soluble polymers,such as polyethylene glycol, (PEG) or polyvinyl pyrrolidone (PVP), withblocks of other biocompatible or biodegradable polymers, for example,poly(lactide), poly(lactide-co-glycolide, or polycaprolcatone orcombinations thereof, polymers and copolymers of ethylene and propylene,including ultra-high molecular weight polyethylene, ultra-high molecularweight polypropylene, nylon, polyesters such as poly(ethyleneterephthalate), poly(tetrafluoroethylene), polyurethanes,poly(ether-urethanes), poly(methylmethacrylate), polyether ether ketone,polyolefins, Dacron, latex, silicones, polymeric cements, andpoly(ethylene oxide).

In another preferred embodiment of the third aspect of the presentinvention, the one or more compounds can be first conjugated with otheragents that have an affinity for, or can react with, a surface, andthereby immobilized on a surface. For example, the compounds can betethered to a linkage that can be photo-activated to bind to a surface,or activated via another mechanism.

Examples of devices and articles that can be coated using thecompositions include tubing and other surface medical devices, such asurinary catheter, stents, mucous extraction catheter, suction catheter,umbilical cannula, contact lenses, intrauterine devices, intravaginaland intraintestinal devices, endotracheal tubes, bronchoscopes, dentalprostheses and orthodontic devices, dentures, teeth, surgicalinstruments, dental instruments, tubing, dental water lines, dentaldrain tubes, fabrics, paper, indicator strips (e.g., paper indicatorstrips or plastic indicator strips), adhesives (e.g., hydrogeladhesives, hot-melt adhesives, or solvent-based adhesives), bandages,tissue dressings or healing devices and occlusive patches, and any othersurface devices used in the medical field. Devices may includeelectrodes, external prostheses, fixation tapes, compression bandages,and monitors of various types. Medical devices also include any devicethat may be placed at the insertion or implantation site such as theskin near the insertion or implantation site, and which include at leastone surface which is susceptible to colonization by biofilm embeddedmicroorganisms. In one specific embodiment, a composition is integratedinto an adhesive, such as tape, thereby providing an adhesive, which mayprevent growth or proliferation of biofilm embedded microorganisms on atleast one surface of the adhesive. Medical devices include surfaces ofequipment in operating rooms, emergency rooms, hospital rooms, clinics,and bathrooms. In a particularly preferred embodiment the followingdevices may be coated with the compounds: catheters, including centralvenous catheters, urinary catheters, dialysis catheters, and indwellingcatheters (for example, catheters for hemodialysis and foradministration of chemotherapeutic agents), cardiac implants includingmechanical heart valves, stents, ventricular assist devices, pacemakers,cardiac rhythm management (CRM) devices, cardiac resynchronizationtherapy devices (CRTs), and implantable cardioverter defibrillators(ICDs), synthetic vascular grafts, arteriovascular shunts, cerebralspinal fluid shunts, cochlear devices, prosthetic joints, orthopedicimplants, internal fixation devices, bone cements, percutaneous sutures,surgical mesh and surgical patches including hernia repair meshes andpatches, breast reconstruction meshes and patches, meshes and patchesfor breast and face lifts, slings, and meshes and patches for pelvicfloor reconstruction, tracheal and ventilator tubing, wound dressings,biological implants (including allografts, xenografts and autografts),penile implants, intrauterine devices, endotracheal tubes, and contactlenses.

Other articles that can be coated in accordance with the third aspect ofthe present invention include articles for use in rearing animals, suchas animals and articles mentioned in the context of the first aspect ofthe present invention.

Yet other articles that can be coated in accordance with the thirdaspect of the present invention include articles for use in the processof slaughter and/or processing the carcasses or parts thereof ofanimals, such as animals and articles mentioned in the context of thefirst aspect of the present invention.

Yet further articles that can be coated in accordance with the thirdaspect of the present invention include articles for the preparationand/or containment of food stuffs, including foodstuffs comprising rawor cooked meats, eggs, dairy products or other food products. The foodproducts may be human and/or animal food products.

Yet further articles that can be coated in accordance with the thirdaspect of the present invention include articles for the preparationand/or containment of drinks.

Accordingly, in another embodiment of the third aspect of the presentinvention there is provided a method of disinfecting a surface, orprotecting a surface against infection, in need thereof, the methodcomprising contacting the surface with an effective amount of one ormore compounds having the structure of having the structure of Formula Aor B, or other compounds of the invention of the present invention asdescribed further in section III.A of this application, wherein the oneor more compounds are coated onto the surface to be disinfected.

In some embodiments the one or more compounds may be applied to thesurface in the form of a spray, an aerosol, or a foam.

The coated surface may, for example, be formed on the surface of aninstrument selected from the group consisting of surgical instruments,cardiac and urinary catheters, implants, and ultrasound probes used insterile body cavities.

The coated surface may, for example, be formed on the surface of adevice selected from the group consisting of urinary catheter, stents,mucous extraction catheter, suction catheter, umbilical cannula, contactlenses, intrauterine devices, intravaginal and intraintestinal devices,endotracheal tubes, bronchoscopes, dental prostheses and orthodonticdevices, surgical instruments, dental instruments, tubing, dental waterlines, dental drain tubes, fabrics, paper, indicator strips (e.g., paperindicator strips or plastic indicator strips), adhesives (e.g., hydrogeladhesives, hot-melt adhesives, or solvent-based adhesives), bandages,tissue dressings or healing devices and occlusive patches, catheters,including central venous catheters, urinary catheters, dialysiscatheters, and indwelling catheters, cardiac implants, mechanical heartvalves, stents, ventricular assist devices, pacemakers, cardiac rhythmmanagement (CRM) devices, cardiac resynchronization therapy devices(CRTs), and implantable cardioverter defibrillators (ICDs), syntheticvascular grafts, arteriovascular shunts, cerebral spinal fluid shunts,cochlear devices, prosthetic joints, orthopedic implants, internalfixation devices, bone cements, percutaneous sutures, surgical mesh andsurgical patches including hernia repair mesh, breast reconstructionmesh, mesh for breast and face lifts, slings, and mesh for pelvic floorreconstruction, tracheal and ventilator tubing, wound dressings,biological implants, penile implants, intrauterine devices, endotrachealtubes, and contact lenses.

The coated surface may, for example, be formed on the surface of anarticle selected from the group consisting of an industrial pipeline,liquid distribution lines, oil and gas pipelines and cosmetic container.

The coated surface may, for example, be formed on the surface of, or beincorporated into, or onto, a household item, such as an item selectedfrom the group consisting of household disinfectants; laundry detergent;cleaning supplies; equipment involved in the leeching process or mining;wound care; toothpaste; mouth wash; dental floss; toothpicks; chewableproducts (including food products); a mouth shield; a dental instrument;dentures; dental retainers; dental braces including plastic braces (suchas Invisalign); bristles of toothbrushes; dental prostheses andorthodontic devices; chewable non-food items, foods, or toys, such asdog bones and biscuits; a vacuum system; HVAC ((heating, ventilation andair conditioning)) systems; vacuum cleaner bags; paint covering; wallcoverings; window frames; doors; door frames; cooling towers;humidifiers; vacuum cleaners; filters such as a vacuum filter, ahumidifier filter, hot tub filter, or a swimming pool filter; toys;plastic bottles; water jugs; tap and water spout; washing machines;dishwashers; animal water dishes; bathroom tiles and fixtures; sinks;showers; shower heads; toilets; toilets lids; toilet seats; sealants andgrout; towels; TUPPERWARE®; dishes; cups; utensils such as forks,spoons, knives, and spatulas; bowls; food storage containers; beveragestorage containers; cutting boards; dish drying trays; garbage bags;sinks; fish ponds; swimming pools; swimming pool liners; swimming poolskimmer; pond liners; bird baths; garden hose; water sprinkling lines;planters; and hot tubs.

The coated surface may, for example, be formed on the surface of, orincorporated into, or onto, an article, device or apparatus used in therearing and/or transport of animals, such as a chicken, for example, ameat-type chicken such as broiler chicken, or an egg-laying chicken suchas a pullet or hen, or a breeder chicken, other poultry, such as aturkey, geese, quail or ducks, or livestock, such as cattle, sheep,goats or swine, alpaca, banteng, bison, camel, cat, deer, dog, donkey,gayal, guinea pig, horse, llama, mule, rabbit, reindeer, water buffalo,yak, although the skilled person will appreciate that other feeds foranimals, including zoo animals, captive animals, game animals, fish(include freshwater and saltwater fish, farmed fish, and ornamentalfish), other marine and aquatic animals, including shellfish such as,but not limited to, oysters, mussels, clams, shrimps, prawns, lobsters,crayfish, crabs, cuttlefish, octopus, and squid, domestic animals suchas cats and dogs, rodents (such as mice, rats, guinea pigs, hamsters),and horses, are also provided, as well as any other domestic, wild andfarmed animal, including mammals, marine animals, amphibians, birds,reptiles, insects and other invertebrates. In some embodiments, thedevice or apparatus used in the rearing and/or transport of animals maybe selected from an article, device or apparatus that is for thedelivery and/or containment of animal feed and/or animal drinking water.

The coated surface may, for example, be formed on the surface of, orincorporated into, or onto, an article, device or apparatus used in therearing, housing and/or transport of animals, such as a chicken, forexample, a meat-type chicken such as broiler chicken, or an egg-layingchicken such as a pullet or hen, or a breeder chicken, other poultry,such as a turkey, geese, quail or ducks, or livestock, such as cattle,sheep, goats or swine, alpaca, banteng, bison, camel, cat, deer, dog,donkey, gayal, guinea pig, horse, llama, mule, rabbit, reindeer, waterbuffalo, yak, although the skilled person will appreciate that otherfeeds for animals, including zoo animals, captive animals, game animals,fish (include freshwater and saltwater fish, farmed fish, and ornamentalfish), other marine and aquatic animals, including shellfish such as,but not limited to, oysters, mussels, clams, shrimps, prawns, lobsters,crayfish, crabs, cuttlefish, octopus, and squid, domestic animals suchas cats and dogs, rodents (such as mice, rats, guinea pigs, hamsters),and horses, are also provided, as well as any other domestic, wild andfarmed animal, including mammals, marine animals, amphibians, birds,reptiles, insects and other invertebrates. In some embodiments, thearticle, device or apparatus used in the rearing, housing and/ortransport of animals can include one or more of an article, device orapparatus used in the production, creation, collection, storage,processing and/or packaging of an animal product. For example, an animalproduct may be a by-product of the animal (e.g. milk, eggs, or wool) ora downstream product thereof. Alternatively, an animal product may bethe body or part of the body of the animal, and the harvesting processoptionally includes the step of slaughtering the animal and furtheroptionally preparing an animal carcass or part thereof as a product,such as a meat product.

The third aspect of the present invention also, therefore, provides adevice, article, product, item, formulation, composition or coating perse, having a coating comprising one or more compounds having thestructure or having the structure of Formula A or B, or other compoundsof the invention of the present invention as described further insection III.A of this application, and for their use in theabove-defined methods.

In one embodiment, the device, article, product, item, formulation,composition or coating comprises the one or more compounds in thecoating in an amount effective to prevent biofilm formation. In anotherembodiment, the device, article, product, item, formulation, compositionor coating comprises the one or more compounds in the coating in anamount effective to treat or reduce biofilm formation.

The third aspect of the present invention also provides the direct perse products of the above-defined methods and uses of the third aspect ofthe present invention, and downstream product produced therefrom.

The third aspect of the present invention also provides a compoundconjugated to a structure that can anchor to a surface, wherein thecompound has the structure of having the structure of Formula A or B, orother compounds of the invention of the present invention as describedfurther in section III.A of this application. It may be preferred thatthe compound is selected from the group consisting of a complex of anamino acid or an α-hydroxy acid with Fe III, such as a complex of quinicacid with Fe III, a complex of L-tyrosine with Fe III, a complex ofL-DOPA with Fe III and/or a complex of L-phenylalanine with Fe III.Optionally, the compound may be selected from the group consisting ofthe compounds represented by Formula I, Formula II, Formula III, FormulaIV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX,Formula X, Formula XI, Formula XII, Formula XIII, Formula XIV, acompound that binds to major outer membrane proteins (MOMPs) or FlaA ofCampylobacter, a synthetic human histo-blood group antigen, a mimetic ofhuman histo-blood group antigen or a synthetic sugar.

Also provided by the third aspect of the present invention is acomposition comprising one or more conjugated compounds as definedabove, and an article coated with one or more of said conjugatedcompounds, or with said composition.

In one embodiment, the structure of the conjugated compound compriseshydroxyapatite or derivative thereof, and the conjugate is capable ofanchoring, or is anchored to, a dental tissue.

For example, in a further embodiment, conjugated forms of the compounds,such as those shown in FIGS. 16A and B wherein the compounds areconjugated to hydroxyapatite may be applied to tooth tissues, such astooth enamel, dentin and pulp in order to prevent dental caries andinfection. In another embodiment, the compounds can be applied usingphoto-reactive chemistry, for example, using conjugated forms of thecompounds such as those shown in FIGS. 15A and B.

4. Industrial, Cosmetic and Consumer Applications

The compositions can be used in accordance with a further embodiment ofthe third aspect of the present invention to disinfect industrialsurfaces, by preventing and/or removing biofilm buildup on suchsurfaces.

In this embodiment, the formation of the biofilm may be prevented orinhibited, or a preformed biofilm may be removed by a method thatcomprises applying a composition of the present invention comprising theone or more compounds having the structure of Formula A or B, or othercompounds of the present invention as described further in section III.Aof this application, onto a surface in need thereof, for example as aspray, foam, gel, powders; dish or laundry detergents (liquid or solid),surface wax, glass cleaner, etc.

Accordingly, the third aspect of the present invention also provides anobject or article that has been treated in accordance with the foregoingmethod.

Biofilms are continuously produced and often accumulate on numerousindustrial surfaces and on biological surfaces. In an industrialsetting, the presence of these biofilms causes a decrease in theefficiency of industrial machinery, requires increased maintenance, andpresents potential health hazards. For example, the surfaces of watercooling towers become increasingly coated with microbially producedbiofilm slime which both constricts water flow and reduces heat exchangecapacity. Water cooling tower biofilms may also harbor pathogenicmicroorganisms such as Legionella pneumophila. Food preparation linesare routinely plagued by biofilm build-up both on the machinery and onthe food product where biofilms often include potential pathogens.Biofilm formation comes with associated problems, such as accelerateddeterioration of equipment through corrosion from cellular byproducts.There may also be a reduction in the efficacy of heat transfer andimpairment of detection devices as the film disrupts transmission.

Pseudomonas aeruginosa readily binds to stainless steel or plastic (e.g.polyvinylchloride, polystyrene) surfaces causing major problems in boththe medical and food industries, forming biofilm. Biofilms readily formon PVC and glass surfaces under the static condition, especially in thefood industry.

a. Industrial Applications

The compositions and coatings in accordance with the third aspect of thepresent invention can be used to clean, or maintain, pipelines and hosesin industries such as food and beverage industries, paper mills, sewagetreatment, drainage, cooling towers and gas and oil industries bycontacting a surface with biofilm growth with the composition.Industrial applications include their use in dairy lines, either as aflush or wash for such lines, or incorporated within the lines, forexample as a coating; liquid distribution lines in the food and beveragemanufacturing or dispensing, for example, use as a coating in feederlines for high sugar or syrup distribution in the manufacturing of softdrinks; pulp and paper mills (for biofouling); in the manufacturing andcontainment of cosmetics from production line equipment down to the endconsumable, either incorporated within the cosmetic or coated on the jarcontaining the cosmetic; in water treatment facilities; in the leachingprocess used in mining; to prevent corrosion caused or accelerated byorganisms, in oil and gas pipelines including fracking pipes, in thesouring of oil fields, in antifouling coatings (for example onsubmarines and boats), and in cooling towers.

b. Consumer and Light Commercial Applications

Consumer and light commercial uses of the compounds and coatings inaccordance with the third aspect of the present invention include theirincorporation in general household disinfectants; laundry detergent;cleaning supplies; equipment involved in the leeching process or mining;wound care; a vacuum system; HVAC (heating, ventilation and airconditioning) systems; vacuum cleaner bags; paint covering; wallcoverings; window frames; doors; door frames; cooling towers; boathulls, humidifiers; vacuum cleaners; filters and membranes, such as avacuum filter, a humidifier filter, hot tub filter, osmosis membranes,or a swimming pool filter; toys; plastic bottles; water jugs;toothpaste, mouthwash, a tap and water spout; incorporation intoplastics for a variety of household items including the inside andoutside of washing machines and dishwashers; animal water dishes;bathroom tiles and fixtures; sinks; showers; shower heads; toilets;toilets lids; toilet seats; sealants and grout; towels; TUPPERWARE®;dishes; cups; utensils such as forks, spoons, knives, and spatulas;bowls; food storage containers; beverage storage containers; cuttingboards; dish drying trays; garbage bags; bathtubs including whirlpooland jacuzzi bathtubs; sinks; fish ponds and tanks; swimming pools;swimming pool liners; swimming pool skimmer; pond liners; bird baths;garden hose; water sprinkling lines; planters; and hot tubs.

c. Cosmetic Applications

A further embodiment of the third aspect of the present inventionprovides cosmetics and cosmetic applications, as well as containers forcosmetics and applicators for cosmetics that incorporate and/or arecoated by, the one or more compounds having the structure of Formula Aor B, or other compounds of the invention of the present invention asdescribed further in section III.A of this application.

Cosmetics (also known as makeup or make-up) include care substances usedto enhance the appearance or odor of the human body. They are generallymixtures of chemical compounds, some being derived from natural sources(including natural oils) and many being synthetics. A cosmetic may be asubstance that is suitable to be applied to the human body forcleansing, beautifying, promoting attractiveness, or altering theappearance without affecting the body's structure or functions. Althoughsoap is traditionally not considered to be a cosmetic, for the purposesof the present description the discussion of cosmetics can also beapplied to soaps.

Exemplary cosmetics include skin-care creams, lotions, powders,perfumes, lipsticks, fingernail and toe nail polish, eye and facialmakeup, towelettes, permanent waves, colored contact lenses, haircolors, hair sprays and gels, deodorants, hand sanitizer, baby products,bath oils, bubble baths, bath salts, butters and many other types ofproducts. A subset of cosmetics is called “make-up,” which refersprimarily to coloring products intended to alter the user's appearance.Cosmetics that are meant to be used on the face and eye area are usuallyapplied with a brush or the fingertips.

Cosmetics may comprise a variety of organic compounds and inorganiccompounds. Typical organic compounds can include modified natural oilsand fats as well as a variety of petrochemically derived agents.Inorganic compounds can include processed minerals such as iron oxides,talc, and zinc oxide. The oxides of zinc and iron may be classified aspigments, i.e. colorants, and may have no solubility in solvents.

The application of the third aspect of the present invention tocosmetics, cosmetic applications, cosmetic containers and/or cosmeticapplicators may provide for methods to reduce, avoid, minimise ordisrupt biofilms in the cosmetics, containers and/or applicators.Further, insofar as the applicant of the cosmetic to the body of theuser achieves the delivery of one or more compounds having the structureof Formula A or B, or other compounds of the invention of the presentinvention as described further in section III.A of this application,then the cosmetics may be used to treat individuals in accordance withany of the embodiments of the second to third aspects of the presentinvention, particularly in the context of treating, reducing, prevent ordisrupting bacterial infections, colonization, or biofilms on the skin,hair, nails, and/or in teeth of the user.

5. Additional Medical Applications

In a further embodiment of the third aspect of the present invention,the compounds having the structure of Formula A or B, or other compoundsof the invention of the present invention as described further insection II.A of this application, and compositions comprising one ormore of said compound, can be used to treat any medical conditionassociated with biofilm formation as a result of microorganismsincluding, but not limited to gram-negative and gram-positive bacteria,including Pseudomonas, H. pylori, E. feacalis, Campylobacter, E. coli,EPEC, UPEC and Staphylococcus.

In addition to the conditions discussed above, rarer, but more seriousmanifestations of MRSA can occur, such as necrotizing fasciitis andpyomyositis (most commonly found in the tropics), necrotizing pneumonia,infective endocarditis (which affects the valves of the heart), and boneand joint infections. Additional conditions include severe or extensivedisease (e.g., involving multiple sites of infection) or rapidprogression in presence of associated cellulitis, signs and symptoms ofsystemic illness, associated comorbidities or immunosuppression,extremes of age, abscess in an area difficult to drain (e.g., face,hand, and genitalia), associated septic phlebitis, and lack of responseto incision and drainage alone, purulent cellulitis, hospitalizedpatients with complicated SSTI (cSSTI; defined as patients with deepersoft-tissue infections, surgical/traumatic wound infection, and infectedulcers and burns), osteomyelitis, device-related osteoarticularinfections.

In a further embodiment, the compounds having the structure of Formula Aor B, or other compounds of the invention of the present invention asdescribed further in section III.A of this application, and compositionscomprising one or more of said compound, may also be used in thetreatment of keratitis, colon cancer (where biofilms play a role), andperi-implantitis, a bacterial infection around an implant that resultsin inflammation of the gums, and can lead to bone loss in the jaw.

Certain strains of enterohaemorrhagic E. coli (EHEC) found in the gut ofboth animals and humans can cause disease, and can be life-threateningin a small group of patients that develop haemolytic uraemic syndrome(HUS). EHEC is not treated with antibiotics because of the risks ofdeveloping HUS. The compounds may be useful in the treatment of EHECinfections both in humans and animals, and particularly in cattle.

Uropathogenic E. coli (UPEC) is the predominant etiologic agent thatcauses UTIs. Accordingly, the compositions can also be used to inhibitor reduce biofilm involved in lower urinary tract infections (UTIs).UTI's in human have been traditionally considered to be a self-limitingdisease involving bacteria residing in the lumen of bladders.Intracellular bacterial community-like structures also have beenidentified in the urine sediments of patients with UTIs in a prospectivestudy.

In one embodiment, the biofilm that is inhibited or disrupted by thethird aspect of the present invention may be a bacterial biofilm. Thebacteria forming the biofilm may be gram positive, or in an alternativeembodiment may be gram negative, or the biofilm may be formed by amixture of gram positive and gram negative bacteria.

Optionally, the biofilm may be formed by bacteria selected from thegroup consisting of S. epidermidis, E. faecalis, E. coli, S. aureus, H.pylori, Campylobacter, Enteropathogenic Escherichia coli (EPEC),Uropathogenic Escherichia coli (UPEC), and Pseudomonas or combinationsthereof. Optionally, in certain embodiments of the third aspect of thepresent invention, the biofilm is a biofilm that is formed by bacteriaother than bacteria that comprise, consist essentially of, or consist ofproteobacteria class, such as any one or more of the spirilloidWolinella spp., Helicobacter spp., and most particularly Campylobacterspp.

Optionally, the one or more compounds administered to a subject (such asa human or animal) in accordance with the third aspect of the presentinvention may be a pharmaceutical or veterinary product, and further mayinclude one or more excipients, such as discussed in section III.C ofthis application, below.

In one embodiment of the third aspect of the present invention, for thetreatment of biofilms in a subject (such as a human or animal), the oneor more compounds is administered to a subject by one or more routesselected from: parenteral delivery, such as discussed below in sectionIII.C.1 of this application, including a controlled release formulation,such as discussed below in section III.C.1(a) of this application, andinjectable or implantable formulation, such as discussed below insection III.C.1(b) of this application; enteral delivery, such asdiscussed below in section III.C.2 of this application, including acontrolled release enteral formulation, such as discussed below insection III.C.2(a) of this application, with further reference toextended release dosage forms and delayed release dosage forms asdiscussed therein; oral delivery; topical delivery, such as discussedbelow in section III.C.3 of this application, including as an emulsion,lotion, cream, ointment, gel, or foam as discussed in parts (a), (b),(c), (d) (e) and (f) respectively below in section III.C.3 of thisapplication; buccal delivery; sublabial delivery; sublingual delivery;in or on a dental product, such as a toothpaste, a mouthwash, a dentalfloss, a mouth shield; dermal delivery; or transdermal delivery.

In some embodiment of the third aspect of the present invention, thebiofilm may be associated with a bacterial infection selected from thegroup consisting of impetigo, boils, abscesses, folliculitis,cellulitis, necrotizing fasciitis, pyomyositis, surgical/traumatic woundinfection, and infected ulcers and burns), osteomyelitis, device-relatedosteoarticular infections, impetigo, secondarily infected skin lesions,meningitis, brain abscess, subdural empyema, spinal epidural abscess,arterial damage, gastritis, urinary tract infections, biliary tractinfections, pyelonephritis, cystitis, sinus infections, ear infections,otitis media, otitis externa, leprosy, tuberculosis, conjunctivitis,bloodstream infections, benign prostatic hyperplasia, chronicprostatitis, lung infections including chronic lung infections of humanswith cystic fibrosis, osteomyelitis, catheter infections, bloodstreaminfections, skin infections, acne, rosacea, dental caries,periodontitis, gingivitis, nosocomial infections, arterial damage,endocarditis, periprosthetic joint infections, open or chronic woundinfections, venous stasis ulcers, diabetic ulcers, arterial leg ulcers,pressure ulcers, endocarditis, pneumonia, orthopedic prosthesis andorthopedic implant infections, peritoneal dialysis peritonitis,cirrhosis, and any other acute or chronic infection that involves orpossesses a biofilm.

A further embodiment of the third aspect of the present inventionprovides a method of treating a microbial infection in a subject in needthereof, the method comprising administering to the subject an effectiveamount of one or more compounds having the structure of Formula A or B,or other compounds of the invention of the present invention asdescribed further in section III.A of this application. Likewise, thisembodiment also provides for the use of one or more of said compoundsfor treating a microbial infection in a subject in need thereof.

In certain embodiments, the microbial infection is caused by bacteria,such as gram positive bacteria, or gram negative bacteria. For example,the infection may be caused by bacteria selected from the groupconsisting of S. epidermidis, E. faecalis, E. coli, S. aureus, H.pylori, Campylobacter, Enteropathogenic Escherichia coli (EPEC),Uropathogenic Escherichia coli (UPEC), and Pseudomonas or combinationsthereof and/or optionally wherein the infection is not caused bybacteria that comprise, consist essentially of, or consist ofproteobacteria class, such as any one or more of the spirilloidWolinella spp., Helicobacter spp., and most particularly Campylobacterspp.

Optionally, in the treatment of a microbial infection in a subject inneed thereof in accordance with this embodiment of the third aspect ofthe present invention, the one or more compounds may be administered toa subject by parenteral delivery; enteral delivery; oral delivery;topical delivery, such as in the form of an emulsion, lotion, cream,ointment, gel or foam; buccal delivery; sublabial delivery; sublingualdelivery; in or on a dental product or dental device, such as a dentalproduct, including but not limited to a toothpaste, a mouthwash, adental floss, toothpicks, chewable products (including food products), amouth shield, a dental instrument, dentures, dental retainers, dentalbraces including plastic braces (such as Invisalign), bristles oftoothbrushes, dental prostheses and orthodontic devices, chewablenon-food items, foods, or toys, such as dog bones and biscuits; dermaldelivery; or transdermal delivery.

In certain embodiments, the treatment of a microbial infection in asubject in need thereof in accordance with the this embodiment of thethird aspect of the present invention may be to treat an infection isselected from the group consisting of impetigo, boils, abscesses,folliculitis, cellulitis, necrotizing fasciitis, pyomyositis,surgical/traumatic wound infection, and infected ulcers and burns),osteomyelitis, device-related osteoarticular infections, impetigo,secondarily infected skin lesions, meningitis, brain abscess, subduralempyema, spinal epidural abscess, arterial damage, gastritis, urinarytract infections, biliary tract infections, pyelonephritis, cystitis,sinus infections, ear infections, otitis media, otitis externa, leprosy,tuberculosis, conjunctivitis, bloodstream infections, benign prostatichyperplasia, chronic prostatitis, lung infections including chronic lunginfections of humans with cystic fibrosis, osteomyelitis, catheterinfections, bloodstream infections, skin infections, acne, rosacea,dental caries, periodontitis, gingivitis, nosocomial infections,arterial damage, endocarditis, periprosthetic joint infections, open orchronic wound infections, venous stasis ulcers, diabetic ulcers,arterial leg ulcers, pressure ulcers, endocarditis, pneumonia,orthopedic prosthesis and orthopedic implant infections, peritonealdialysis peritonitis, cirrhosis, and any other acute or chronicinfection that involves or possesses a biofilm.

In certain embodiments for the treatment of a microbial infection in asubject in need thereof in accordance with this embodiment of the thirdaspect of the present invention, the infection may be caused by adrug-resistant strain of E. coli.

Optionally, the treatment of a microbial infection in a subject in needthereof in accordance with this embodiment of the third aspect of thepresent invention may be for the treatment of a urinary tract infection.

Optionally, the treatment of a microbial infection in a subject in needthereof in accordance with this embodiment of the third aspect of thepresent invention, the subject may be one that is hospitalized and/or isimmunocompromised.

Optionally, the treatment of a microbial infection in a subject in needthereof in accordance with this embodiment of the third aspect of thepresent invention may also include further administering one or moreantimicrobial agents, such as one or more antibiotics, to the subject.This may, for example, be conducted in accordance with any one or moreof the embodiments of the second aspect of the present invention.

III. Compounds and Compositions

The present inventors have identified a class of a broad range ofactivity, particularly against bacteria, and has developed numerous usesfor, and methods involving, the compounds, particularly in the formationof compositions. The compounds, which are further defined in SectionIII.A of this application, below, and compositions comprising one ormore of said compounds, are presented herewith as a fourth aspect of thepresent invention. The compounds and compositions comprising one or moreof the compounds can be used to inhibit or reduce biofilm formation on asurface, treat or prevent an infection, and kill some antibioticresistant organisms. In one embodiment, the invention is generallydirected to compounds and compositions comprising one or more of thecompounds, and methods and uses employing one or more of the compoundsand/or compositions, for inhibiting, reducing, or preventing biofilmformation or buildup on a surface or to removing, dispersing, reducing,or eradicating biofilm on a surface. In another embodiment, theinvention also generally relates to compounds and compositionscomprising one or more of the compounds, and methods and uses employingone or more of the compounds and/or compositions, for the treatment of,inhibition of growth of, and inhibition of colonization by, bacteria,both in biological and non-biological environments. In a furtherembodiment, the invention also relates to compounds and compositionscomprising one or more of the compounds, and methods and uses employingone or more of the compounds and/or compositions, for disinfectingsurfaces, both in biological and non-biological environments, andproducts that have been coated with, or treated by, one or more of thecompounds and/or compositions of the present invention. In anotherembodiment, the invention also relates to compounds and compositionscomprising one or more of the compounds, and methods and uses employingone or more of the compounds and/or compositions, for potentiating theeffects of one or more antibiotics, increasing the sensitivity ofbacteria (including antibiotic-resistant bacteria) to one or moreantibiotics, and also to reversing antibiotic resistance in bacteria. Inyet another embodiment, the invention also relates to compounds andcompositions comprising one or more of the compounds, and methods anduses employing one or more of the compounds and/or compositions, forenhancing the growth of animals and their efficiency of feedutilization, in particular by oral administration of feed and drinkcompositions.

A. Compounds

The following compounds as described in this section of the applicationare provided herewith as a fourth aspect of the present invention.

All other aspects of the present invention may utilize one or more typesof compounds as defined in this section, including derivatives and saltsas defined in sub-sections 1 and 2, respectively.

Compositions comprising, consisting essentially of, or consisting of,one or more of these compounds is also provided as a further embodimentof the fourth aspect of the present invention. These compositions may beused in all of the other various aspects of the present invention, andmethods and uses of the present invention which employ saidcompositions, and may comprise, consist essentially of, or consist of,one or more types of compound as defined in this section, includingderivatives and salts as defined in sub-sections 1 and 2, respectively.

Without limitation, compounds of particular interest for use inaccordance with the present invention include Fe III complexescomprising ligands bound to the iron centre selected from amino acids orα-hydroxy acids, including but not limited to ferric quinate (alsoreferred to herein interchangeable as FeQ and Fe-QA), ferric tyrosine(also referred to herein as FeTyr), ferric DOPA (also referred to hereinas FeDOPA), and ferric phenylalanine (also referred to herein asFe-Phe). Further, compounds which are structural and/or functionalvariants, derivatives and/or analogs of the foregoing compounds, asfurther described below in this section, are of particular interest tothe present invention.

The ligands that may be used in such complexes include ligands based onamino acids, α-hydroxy acids, o-hydroxy benzoic acids orpyridine-2-carboxylic acids.

Exemplary amino acids can include, but are not limited to alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline, each preferably in the L-isoform although, as discussed above,in an alternative embodiment one or more (optionally all) may be in theD-isoform. Mixtures of optical isomers of the same amino acid may, ormay not, be used in some embodiments.

Exemplary α-hydroxy acids include, but are not limited to, quinic acid,lactic acid, glycolic acid, citric acid and mandelic acid.

Exemplary o-hydroxy benzoic acids include, but are not limited to,salicylic acid.

Exemplary pyridine-2-carboxylic acids include, but are not limited to,α-Picolinic acid.

In certain embodiments, compounds for use in the present invention, andwhich may bind to MOMPs or FlaA of Campylobacter, are Fe III complexeseach containing three bidentate ligands, such as described herein.

In further embodiments, compounds for use in the present invention, andwhich may optionally bind to MOMPs or FlaA of Campylobacter, are Fe IIIcomplexes defined according to the following chemical Formula A:

or a salt and/or hydrate thereof, wherein:

X, X¹ and X² can independently be NH₂, OH, CO₂—, CO₂H, OR³, NR³H, NR³R⁴,R³ONO₂, R³NO₂, SH, SR³, and X, X¹ and X² may all be the same or they mayall be different, or, alternatively, two may be the same and one may bedifferent;

Y, Y¹ and Y² can independently be O, NH, NH₂, NR³, NR³R⁴, SH, OR³, OH,and Y, Y¹ and Y² may all be the same or they may all be different, or,alternatively, two may be the same and one may be different;

Z, Z¹ and Z² may independently be: O, S, NH, NR³, and Z, Z¹ and Z² mayall be the same or they may all be different, or, alternatively, two maybe the same and one may be different;

R, R′, R¹, R^(1′), R², and R^(2′) can independently be H, CH₃, CH₂SH,CH₂CO₂H, CH₂CH₂CO₂H, CH₂C₆H₅, CH₂C₃H₃N₂, CH(CH₃)CH₂CH₃, (CH₂)₄NH₂,CH₂CH(CH₃)₂, CH₂CH₂SCH₃, CH₂CONH₂, (CH₂)₄NHCOC₄H₅NCH₃, CH₂CH₂CH₂,CH₂CH₂CONH₂, (CH₂)₃NHC(NH)NH₂, CH₂OH, CH(OH)CH₃, CH₂SeH, CH(CH₃)₂,CH₂C₈H₆N, CH₂C₆H₄OH and R, R′, R¹, R^(1′), R², and R^(2′) may all be thesame or they may all be different, or, alternatively, up to five may bethe same and one or more may be different; or

any relevant pair of R and R′, R¹ and R^(1′), and R² and R^(2′) (i.e.when they are bound to the same carbon atom) are linked to form asubstituted or unsubstituted cycloalkyl ring group;

R³ and R⁴ can independently be alkyl, alkenyl, alkynyl, phenyl, aryl,halo- and hydroxy-substituted radicals, hydroxyl radicals,nitrogen-substituted radicals, oxygen-substituted radicals, or hydrogen.In some embodiments, R³ and R⁴ may all be the same or they may all bedifferent, or, alternatively, two may be the same and one may bedifferent.

In embodiments in which one or more pairs of R and R′, R¹ and R^(1′),and R² and R^(2′) are linked to form a substituted or unsubstitutedcycloalkyl ring group, the substituents on the cycloalkyl group can beselected from, but are not limited to, ═O and, particularly, OH, NH₂,NR³, NR³R⁴, SH, and OR³; where R³ and R⁴ are as defined above.

It is preferred that the bonds between the Fe and X, X¹ and X² andbetween the Fe and Y, Y¹ and Y² are ionic.

In a particular embodiment, X, X¹ and X² can independently be NH₂, OH,CO₂—, CO₂H, OR³, NR³H or NR³R⁴ (preferably NH₂ or OH);

Y, Y¹ and Y² can independently be O, NH, NH₂, OR³ or OH (preferably O);

Z, Z¹ and Z² may independently be O or S (preferably O);

R, R′, R¹, R^(1′), R², and R^(2′) can independently be H, CH₃, CH₂SH,CH₂CO₂H, CH₂CH₂CO₂H, CH₂C₆H₅, CH₂C₃H₃N₂, CH(CH₃)CH₂CH₃, (CH₂)₄NH₂,CH₂CH(CH₃)₂, CH₂CH₂SCH₃, CH₂CONH₂, (CH₂)₄NHCOC₄H₅NCH₃, CH₂CH₂CH₂,CH₂CH₂CONH₂, (CH₂)₃NHC(NH)NH₂, CH₂OH, CH(OH)CH₃, CH₂SeH, CH(CH₃)₂,CH₂C₈H₆N, CH₂C₆H₄OH; or

any relevant pair of R and R′, R¹ and R^(1′), and R² and R^(2′) arelinked to form a 4- to 6-membered substituted or unsubstitutedcycloalkyl ring group (optionally wherein the substituents on thecycloalkyl group are selected from ═O and, particularly, OH, NH₂, NHR³,NR³R⁴, SH, and OR³); and

R³ and R⁴ independently represent methyl, ethyl, propyl, butyl, orbenzyl.

Particular compounds that may be mentioned include those in which R′,R^(1′) and R^(2′) represent H, and R, R¹ and R² represent a group asdefined above other than H; or

each pair of R and R′, R¹ and R^(1′), and R² and R^(2′) (i.e. when theyare bound to the same carbon atom) are linked to form a cyclohexyl ringgroup optionally substituted one or more substituents selected from ═Oand, particularly, OH, NH₂, NHR³, NR³R⁴, SH, and OR³.

In a further embodiment, R³ and R⁴ are independently C₁₋₄ alkyl, C₁₋₄alkenyl, phenyl or benzyl (which latter four groups are optionallysubstituted by one or more halo or hydroxyl groups). For example, R³ andR⁴ may independently represent methyl, ethyl, propyl, butyl or benzyl.

In a preferred embodiment, Y, Y¹ and Y² represent O, and Z, Z¹ and Z²represent O. Particular examples of such compounds include those inwhich X, X¹ and X² independently represent NH₂ or OH.

Functional variants of compounds according to Formula A may also be usedin the present invention, and include other compounds as described inthis section of the application.

For example, in a yet further embodiment, compounds for use in thepresent invention, and which may bind to MOMPs or FlaA of Campylobacter,are Fe III complexes defined according to the following chemical FormulaB:

or a salt and/or hydrate thereof, wherein:

X³, X⁴ and X⁵ can independently be —C(R⁸)═, or —N═;

R⁸ can independently be NH₂, OH, CO₂—, CO₂H, OR⁹, NR⁹H, NR⁹R¹⁰, R⁹ONO₂,R⁹NO₂, SH, SR⁹, and each R⁸ may all be the same or they may all bedifferent, or, alternatively, two may be the same and one may bedifferent;

Y³, Y⁴ and Y⁵ can independently be O, NH, NH₂, NR⁹, NR⁹R¹⁰, SH, OR⁹, OH,and Y³, Y⁴ and Y⁵ may all be the same or they may all be different, or,alternatively, two may be the same and one may be different;

Z³, Z⁴ and Z⁵ may independently be: O, S, NH, NR⁹, and Z³, Z⁴ and Z⁵ mayall be the same or they may all be different, or, alternatively, two maybe the same and one may be different;

m1, m2 and m3 may independently be 0, 1, 2, 3 or 4; and m1, m2 and m3may all be the same or they may all be different, or, alternatively, twomay be the same and one may be different;

R⁵, R⁶ and R⁷ are each independently selected from OH, NH₂, NHR⁹,NR⁹R¹⁰, SH, and OR⁹; and R⁵, R⁶ and R⁷ may all be the same or they mayall be different;

R⁹ and R¹⁰ can independently be alkyl, alkenyl, alkynyl, phenyl, aryl,halo- and hydroxy-substituted radicals, hydroxyl radicals,nitrogen-substituted radicals, oxygen-substituted radicals, or hydrogen.In some embodiments, R⁹ and R¹⁰ may all be the same or they may all bedifferent.

It is preferred that the bonds between the Fe and X³, X⁴ and X⁵ andbetween the Fe and Y³, Y⁴ and Y⁵ are ionic.

In a particular embodiment, X³, X⁴ and X⁵ can independently be —C(OH)═,or —N═;

Y³, Y⁴ and Y⁵ can independently be O, NH, NH₂, OR⁹ or OH (preferably O);

Z, Z¹ and Z² may independently be O or S (preferably O);

R⁵, R⁶ and R⁷ are each independently selected from OH, NH₂, NHR⁹, andOR⁹ (preferably R⁵, R⁶ and R⁷ are all OH);

m1, m2 and m3 may independently be selected from 0, 1 and 2; and

R⁹ and R¹⁰ independently represent methyl, ethyl, propyl, butyl, orbenzyl.

In a further embodiment, R⁹ and R¹⁰ are independently C₁₋₄ alkyl, C₁₋₄alkenyl, phenyl or benzyl (which latter four groups are optionallysubstituted by one or more halo or hydroxyl groups). For example, R³ andR⁴ may independently represent methyl, ethyl, propyl, butyl or benzyl.

In a preferred embodiment, Y³, Y⁴ and Y⁵ represent O, Z³, Z⁴ and Z⁵represent O, R⁵, R⁶ and R⁷ represent OH, and m1, m2 and m3 are selectedfrom 0, 1 and 2. Particular examples of such compounds include those inwhich X³, X⁴ and X⁵ independently represent —C(OH)═ or —N═.

In a further preferred embodiment, the ligands bound to the iron centreare amino acids or α-hydroxy acids. Therefore, it is most preferred thatY, Y¹, Y², Z, Z¹ and Z² represent O, X, X¹ and X² represent NH₂ or OH,and R′, R^(1′) and R^(2′) represent H. Where one or more of the ligandsis an amino acid (e.g. for compounds of formula A in which X, X¹ and X²represent NH₂), then it is preferred that the amino acid is an L-aminoacid (or glycine), although in an alternative embodiment one or more(optionally all) of the ligands may be a D-amino acid. Exemplary aminoacids can include, but are not limited to alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine, each preferably in theL-isoform although, as discussed above, in an alternative embodiment oneor more (optionally all) may be in the D-isoform. Mixtures of opticalisomers of the same amino acid may, or may not, be used in someembodiments.

Exemplary compounds of Fe complexes according to Formula A includeFormulas VII-IX as shown below:

g) a complex of L-DOPA with Fe III (3,4 dihydrophenylalanine) (Fe-DOPA)

h) a complex of L-tyrosine with Fe III (Fe-Tyr, also denoted Fe—Y)

i) a complex of quinic acid with Fe III (Fe-QA, also denoted FeQ)

Exemplary compounds of Fe complexes according to Formula B includeFormulas X-XIV as shown below:

j) a complex of 2,3,5-trihydroxybenzoic acid with Fe III

k) a complex of 2,4,5-trihydroxybenzoic acid with Fe

l) a complex of 3-dehydroquinic acid with Fe III

m) a complex of 4,6-dihydroxypyridine-2-carboxylic acid with Fe III

n) a complex of salicylic acid with Fe III

Optionally, in one embodiment, an Fe complex as described above (e.g.according to Formula A or Formula B) for use in any of the first, secondor third aspects of the present invention may not be a complex of quinicacid with Fe III (such as a complex having the structure of Formula IX).That is to say, in one optional embodiment, Formula A excludes a complexof quinic acid with Fe III (such as a complex having the structure ofFormula IX).

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound that inhibits thebinding of C. jejuni to a histo-blood group antigen. This can, forexample, be measured when the bacteria is grown in a medium containingthe compound, the medium containing the compound is washed away, and thebinding of the bacteria to the histo-blood group antigen is determinedby an ELISA assay (such as in accordance with the method as described inExample 4) and compared to a control where the bacteria is not grown inthe presence of the compound. Preferably the compound inhibits thebinding of C. jejuni to a histo-blood group antigen at a level that isat, or at least, about 1%, 2%, 3%, 4%, more preferably at, or at least,about 5%, even more preferably at, or at least, about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more thanthe level of inhibition of the binding of C. jejuni to a histo-bloodgroup antigen by either a complex of L-tyrosine with Fe III or a complexof quinic acid with Fe III at the same molar concentration.

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound that inhibitsbiofilm formation by bacteria as measured in a plastic bead assay (suchas in accordance with a method as described in Example 1), wherein thebacteria is grown in a medium containing the compound to form a growthsuspension of the bacteria at 0.0001 OD/ml, the growth suspension isallowed to grow with plastic coated UV beads (Lascells), and the beadsare assayed after 24 hours for the presence of biofilm formation on thebeads (by counting bacteria after release from the beads), and comparedto a control group where the bacteria is not grown in the presence ofthe compound. Preferably the compound inhibits the binding of thebacteria to the plastic coated beads at a level of inhibition that isat, or at least, about 1%, 2%, 3%, 4%, more preferably at, or at least,about 5%, even more preferably at, or at least, about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more ofthe level of inhibition of the binding of the bacteria to the plasticcoated UV beads by either a complex of L-tyrosine with Fe III or acomplex of quinic acid with Fe III at the same molar concentration. Inparticularly preferred embodiment, the bacteria can be Enterococcusfaecalis, Staphylococcus epidermidis, Staphylococcus aureus,Campylobacter jejuni, Pseudomonas aeruginosa, Uropathogenic Escherichiacoli, and Enteropathogenic Escherichia coli.

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound that inhibitsbinding of Helicobacter pylori to human gastric tissue (for example asdetermined by a method as described in Example 5) at a level ofinhibition that is at, or at least, about 1%, 2%, 3%, 4%, morepreferably at, or at least, about 5%, even more preferably at, or atleast, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 100% or more of the level of inhibition of the binding of thebacteria to human gastric tissue by either a complex of L-tyrosine withFe III or a complex of quinic acid with Fe III at the same molarconcentration as measured by counting the average number of bacteriabound to the tissue.

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound that inhibitsbiofilm formation of a bacteria, but does not inhibit planktonic growthof the bacteria (for example, as determined using a method as describedin Example 7), wherein the bacteria can be one or more of the following:Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcusaureus, Campylobacter jejuni, Pseudomonas aeruginosa, UropathogenicEscherichia coli, and Enteropathogenic Escherichia coli. Preferably thecompounds inhibit biofilm formation (for example, as measured bycoverage rate in Example 7), at a level that is at, or at least, about1%, 2%, 3%, 4%, more preferably at, or at least, about 5%, even morepreferably at, or at least, about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the level of biofilminhibition by a complex of L-tyrosine with Fe III or a complex of quinicacid with Fe III at the same molar concentration.

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound for the treatmentof cystic fibrosis. In one embodiment, one or more compounds of FormulaA may be delivered using a nebulizer spray. In another embodiment, oneor more compounds of Formula A may be delivered in liposomes for thetreatment of patients with cystic fibrosis.

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound that preventsattachment of bacteria to a surface (for example, when determined inaccordance with a method as described in Example 13), and the preventionof attachment of bacteria to the surface is at a level that is at, or atleast, about 1%, 2%, 3%, 4%, more preferably at, or at least, about 5%,even more preferably at, or at least, about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the levelof bacteria attachment by a complex of L-tyrosine with Fe III or acomplex of quinic acid with Fe III at the same molar concentration asmeasured by optical density. In particularly preferred embodiment, thebacteria can be Enterococcus faecalis, Staphylococcus epidermidis,Staphylococcus aureus, Campylobacter jejuni, Pseudomonas aeruginosa,Uropathogenic Escherichia coli, and Enteropathogenic Escherichia coli.

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound that is capable ofrendering an antibiotic resistant strain of bacteria sensitive to theantibiotic to which it is otherwise resistant (for example, whendetermined by a method that comprises immersing a patch in a solution ofthe compound and an antibiotic, such as kanamycin, for example at aconcentration of 50 μg/mL as described in Example 9, placed on a platewith the antibiotic resistant strain (such as a kanamycin resistantstrain of Enteropathogenic Escherichia coli or Campylobacter jejuni)),and causes the bacteria to fail to grow or reduces the rate of growth ofthe antibiotic resistant strain in the presence of the antibiotic by alevel that is a level that is at, or at least, about 1%, 2%, 3%, 4%,more preferably at, or at least, about 5%, even more preferably at, orat least, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, 100% or more of the level of reduction of the rate ofgrowth caused by a complex of L-tyrosine with Fe III or a complex ofquinic acid with Fe III at the same molar concentration.

In a further embodiment, a compound according to Formula A, or FormulaB, for use in the present invention may be a compound that causes adecrease in the rate of growth to a level that is at, or at least, about1%, 2%, 3%, 4%, more preferably at, or at least, about 5%, even morepreferably at, or at least, about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more of the decrease in therate of growth measured by optical density of an antibiotic resistantbacteria when grow in the presence of the compound and the antibiotic,for example as determined by a method as described in Examples 11 and/or12. The combinations of antibiotics and antibiotic resistant bacteriacan, for example, be one or more of the following: (i) kanamycin and akanamycin-resistant bacteria, (ii) gentamicin and a gentamicin-resistantenteropathogenic Escherichia coli, and (iii) kanamycin and a clinicalisolate of Pseudomonas (PAO Clinical) as described in Example 14.

In accordance with one embodiment, instead of the direct administrationof the one or more compounds, it or they may be formed in vivo, byadministering a suitable iron containing substance and one or moresuitable ligands capable of forming the compounds in vivo with the ironcompound (see: Campbell and Hasinoff Ferrous sulfate reduces levodopabioavailability: Chelation as a possible mechanism, Clin. Pharmacol.Ther. 45:220-5, 1989). For example, ferrous sulfate and tyrosine (asligand) may be administered in order to form Fe-Tyr in vivo, ferroussulfate and L-DOPA (as ligand) may be administered in order to formFe-DOPA in vivo, ferrous sulfate and L-phenylalanine (as ligand) may beadministered in order to form Fe-Phe in vivo or ferrous sulfate andquinic acid (as ligand) may be administered in order to from Fe-QA invivo. In this example, Fe²⁺ is oxidized to Fe³⁺ in vivo, and may complexwith tyrosine, L-DOPA, or phenylalanine respectively. The compounds mayalso be formed in vivo from any substance that can be metabolized invivo to the compounds. For example, phenylalanine could be administeredwith ferrous sulfate since it will be metabolized to tyrosine in vivo,and may then complex with the ferric iron (formed from oxidation offerrous sulfate). Alternatively, ferric chloride could also beadministered with, for example, tyrosine, quinic acid, L-DOPA and/orphenylalanine.

Optionally, one or more compounds for use in any of the first, second orthird aspects of the present invention (which may or may not becompounds according to Formula A or Formula B as discussed above) areligands for the major outer membrane proteins (MOMPs) or FlaA ofCampylobacter, and/or may be capable of downregulating the expression ofFlaA and/or FlaB proteins in a bacteria such as Campylobacter, such asto the extent of causing a reduced bacterial motility such as whendetermined by a method as described in Example 21 of the presentapplication. The binding of the compounds to the MOMPs or FlaA inhibitsthe MOMPs or FlaA from attaching, binding, or associating with otherproteins, biofilm components, surfaces or other bacteria.

The compound can be a mimetic or synthetic human histo-blood groupantigen or a synthetic sugar. A synthetic human histo-blood groupantigen may be a sugar, for example a saccharide having the samestructure as a natural human histo-blood group antigen such as forexample H-I antigen, H-II antigen, Lewis antigen, Le^(b), Le^(x) orLe^(y). A preferred compound is ferric quinate (Fe-QA).

The compounds provided herein which bind to MOMPs or FlaA ofCampylobacter include compounds with structures described in thissection, in accordance with Formulae A or B, or further compounds asdescribed below. It has been demonstrated that these compounds inhibitboth gram negative bacteria, such as Pseudomonas aeruginosa,Campylobacter jejuni, Helicobacter pylori, Escherichia coli,Enteropathogenic Escherichia coli (EPEC), Uropathogenic Escherichia coli(UPEC) and gram positive bacteria, such as Staphylococcus epidermidis,Staphylococcus aureus, and Enterococcus faecalis, which are believed tobe predictive of efficacy with other species There is low homologybetween the MOMP of Campylobacter and other bacteria. It is believedthat the compounds interact with several surface porin-like bacterialproteins that have not yet been identified on other bacteria.

In further embodiments, compounds for use in the present invention may,or may not, optionally include one or more compounds selected from:

a) N-[³-quinylamino-2-(quinylaminomethyl)-propyl]-quinamide

b) N-{2-[Bis-(2-quinylaminoethyl)-amino]-ethyl}-quinamide

c) Phosphoric acid tris-(2-quinylamino-ethyl) ester

d) N-(3,5-Bis-quinylamino-cyclohexyl)-quinamide

e)N-(4,5-Bis-quinylamino-2-hydroxy-6-hydroxymethyl-tetrahydropyran-3-yl)-quinamide

f)N-(4,5-Bis-quinylamino-2-hydroxy-6-quinylaminomethyl-tetrahydropyran-3-yl)-quinamide

The foregoing compounds for use in any of the aspects of the presentinvention may also be in the form of hydrates, or salts of hydrates. Forexample, the compositions may be Fe-Tyr.xH₂O, FeQ.xH₂O, FeDOPA.xH₂O orFe-Phe.xH₂O. The compounds may also be hydrates containing salts, forexample hydrates with bases such as lithium hydroxide, sodium hydroxideor potassium hydroxide present.

In the case of compounds which are Fe III complexes comprising ligandsbound to the iron centre, as described above, in one option not allligands will be the same in the compositions comprising the Fe IIIcomplex compounds. For example, in the case that the compound is FeTyr,then this may be formed by creating a complex from Fe III and acommercial source of tyrosine (Tyr), which may include low levels(typically, less than 10%, such as less than 5% or about 2.5%) of one ormore further amino acids, such as cysteine (Cys) and/or phenylalanine(Phe), and so in one optional embodiment, when the compound is FeTyr,then some of the compounds in the composition may include one or morealternative amino acids (e.g. Cys and/or Phe) as ligands. The proportionof ligands in the FeTyr composition that are not Tyr may be less than10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% and may be substantially 0%.The same applies mutatis mutandis to other ligands used in thepreparation of Fe III complexes for use in the present invention.

Therefore, for example, in a composition comprising an Fe III complex asdescribed above, it may be that less than 100% of the Fe III ligands areidentical, although preferably at least 50%, 60%, 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the ligands in thecomposition are identical. In that context, in one embodiment the term“identical” discriminates between enantiomeric forms of ligand, that is,different enantiomers are not identical; whereas, in another embodiment,the term “identical” can be applied to different enantiomeric forms ofligand, that is, optionally different enantiomeric forms of the sameligand are considered to be identical.

1. Derivatives

Derivatives of the compounds for use in accordance with any of theaspects of the present invention, such as the compounds defined above,including Formula I-IX, Formula X to XIV, Formula A (or hydratesthereof) and Formula B or hydrates thereof), may also be used. The term“derivative” does not mean that the derivative is synthesized from theparent compound either as a starting material or intermediate, althoughthis may be the case. The term “derivative” can include salts (forexample, pharmaceutically acceptable salts), prodrugs, or metabolites ofthe parent compound. Derivatives include compounds in which free aminogroups in the parent compound have been derivatized to form aminehydrochlorides, p-toluene sulfoamides, benzoxycarboamides,t-butyloxycarboamides, thiourethane-type derivatives,trifluoroacetylamides, chloroacetylamides, or formamides. Derivativesinclude compounds having one or more amino substituents or hydrogengroups replaced with substituted or unsubstituted alkyl, aminoalkyl,aryl, or heteroaryl groups having from 1 to 30 carbon atoms.

2. Salts

The compounds for use in accordance with any of the aspects of thepresent invention, such as the compounds defined above, including ofFormula I-IX, Formula X to XIV, Formula A (or hydrates thereof) andFormula B or hydrates thereof) can be in the form of a salt, forexample, a pharmaceutically acceptable salt. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; andalkali or organic salts of acidic residues such as carboxylic acids. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids and inorganic ororganic bases. Such conventional non-toxic salts include those derivedfrom inorganic acids such as hydrochloric, hydrobromic, sulfuric,sulfamic, phosphoric, and nitric acids; and the salts prepared fromorganic acids such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, tolunesulfonic, naphthalenesulfonic,methanesulfonic, ethane disulfonic, oxalic, and isethionic salts, andbases such as lithium hydroxide, sodium hydroxide, potassium hydroxideand ammonium hydroxide.

The pharmaceutically acceptable salts of the compounds can besynthesized from the parent compound, which contains a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704; and “Handbook of Pharmaceutical Salts:Properties, Selection, and Use,” P. Heinrich Stahl and Camille G.Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

B. Antimicrobial Agents

Antimicrobial agents that may be used therapeutically and/ornon-therapeutically with the compounds of the present invention inaccordance with any of the first, second, or third aspects of thepresent invention, for example for the treatment or prophylaxis ofmicrobial infection in accordance with the third aspect of the presentinvention and/or in accordance with the second aspect of the presentinvention, either separately, simultaneously or sequentially, include,but are not limited to: (i) Aminoglycosides, including amikacin,gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin,streptomycin, spectinomycin; (ii) Ansaycins, including geldanamycin,herbimycin, rifaximin, (iii) Carbacephem, including loracarbef, (iv)Carbapenems, including ertapenem, doripenem, imipenem/cilastatin,meropenem, (v) Cephalosporins, including cefadroxil, cefazolin,cefalotin or cefalothin, cephalexin, cefaclor, cefamandole, cefoxitin,cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone,cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, (vi)Glycopeptides, including teicoplanin, vancomycin, telavancin,dalbavancin, oritavancin, (vii) Lincosamides, including clindamycin,lincomycin, (viii) Lipopeptides including daptomycin, (ix) Macrolidesincluding azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spiramyin, (x)Monobactams, including aztreonam, (xi) Nitrofurans, includingfurazolidone, nitrofurantoin, (xii) Oxazolidinones, including linezolid,posizolid, radezolid, torezolid, (xiii) Penicillins, includingamoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin,oxacillin, penicillin G, penicillin V, piperacillin, temocillin,ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam,peperacillin/tazobactam, ticarcillin/clavulanate (xiv) Polypeptidesincluding bacitracin, colistin, polymyxin B, (xv)Quinolones/Fluoroquinolone, including ciprofloxacin, enoxacin,gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,sparfloxacin, temafloxacin, (xvi) Sulfonamides, including mafenide,sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine,sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine,sulfisoxazole, trimethoprim-sulfamethoxazole(co-trimoxazaole),sulfonamidochrysoidine, (xvii) Tetracyclines, including demeclocycline,doxycycline, minocycline, oxytetracycline, tetracycline, (xviii)clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide,isoniazid, pyrazinamide, rifampicin (rifampin), rifabutin, rifapentine,streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid,metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin,thiamphenicol, tigecycline, tinidazole, and trimethoprim; andcombinations thereof. The compounds may also be combined with triclosanand chlorhexidine. Other antimicrobial agents include: aztreonam;cefotetan and its disodium salt; loracarbef; cefoxitin and its sodiumsalt; cefazolin and its sodium salt; cefaclor; ceftibuten and its sodiumsalt; ceftizoxime; ceftizoxime sodium salt; cefoperazone and its sodiumsalt; cefuroxime and its sodium salt; cefuroxime axetil; cefprozil;ceftazidime; cefotaxime and its sodium salt; cefadroxil; ceftazidime andits sodium salt; cephalexin; cefamandole nafate; cefepime and itshydrochloride, sulfate, and phosphate salt; cefdinir and its sodiumsalt; ceftriaxone and its sodium salt; cefixime and its sodium salt;cefpodoxime proxetil; meropenem and its sodium salt; imipenem and itssodium salt; cilastatin and its sodium salt; azithromycin;clarithromycin; dirithromycin; erythromycin and hydrochloride, sulfate,or phosphate salts, ethylsuccinate, and stearate forms thereof,clindamycin; clindamycin hydrochloride, sulfate, or phosphate salt;lincomycin and hydrochloride, sulfate, or phosphate salt thereof,tobramycin and its hydrochloride, sulfate, or phosphate salt;streptomycin and its hydrochloride, sulfate, or phosphate salt; neomycinand its hydrochloride, sulfate, or phosphate salt; acetyl sulfisoxazole;colistimethate and its sodium salt; quinupristin; dalfopristin;amoxicillin; ampicillin and its sodium salt; clavulanic acid and itssodium or potassium salt; penicillin G; penicillin G benzathine, orprocaine salt; penicillin G sodium or potassium salt; carbenicillin andits disodium or indanyl disodium salt; piperacillin and its sodium salt;ticarcillin and its disodium salt; sulbactam and its sodium salt;moxifloxacin; ciprofloxacin; ofloxacin; levofloxacins; norfloxacin;gatifloxacin; trovafloxacin mesylate; alatrofloxacin mesylate;trimethoprim; sulfamethoxazole; demeclocycline and its hydrochloride,sulfate, or phosphate salt; doxycycline and its hydrochloride, sulfate,or phosphate salt; oxytetracycline and its hydrochloride, sulfate, orphosphate salt; chlortetracycline and its hydrochloride, sulfate, orphosphate salt; metronidazole; dapsone; atovaquone; rifabutin;linezolide; polymyxin B and its hydrochloride, sulfate, or phosphatesalt; sulfacetamide and its sodium salt; clarithromycin; and silverions, salts, and complexes.

One preferred embodiment of any of the aspects of the present invention,such as in accordance with the second aspect of the present invention,envisages the use of a complex of quinic acid with Fe III (Fe-QA, alsodenoted FeQ), such as defined by Formula IX, with any one or more of theforegoing antibiotics, either formulated together in the samecomposition for administration or presented in separate compositions foruse separately, simultaneously or sequentially.

Another preferred embodiment, of any of the aspects of the presentinvention, such as in accordance with the second aspect of the presentinvention, envisages the use of a complex of L-tyrosine with Fe III(Fe-Tyr), such as defined by Formula VIII, with any one or more of theforegoing antibiotics, either formulated together in the samecomposition for administration or presented in separate compositions foruse separately, simultaneously or sequentially.

In another preferred embodiment, of any of the aspects of the presentinvention, such as in accordance with the second aspect of the presentinvention, envisages the use of a complex of L-DOPA with Fe III (3,4dihydrophenylalanine) (Fe-DOPA), such as defined by Formula VII, withany one or more of the foregoing antibiotics, either formulated togetherin the same composition for administration or presented in separatecompositions for use separately, simultaneously or sequentially.

In another preferred embodiment, of any of the aspects of the presentinvention, such as in accordance with the second aspect of the presentinvention, envisages the use of a complex of L-phenylalanine with Fe III(Fe-Phe), with any one or more of the foregoing antibiotics, eitherformulated together in the same composition for administration orpresented in separate compositions for use separately, simultaneously orsequentially.

C. Excipients and Carriers

The compounds as defined in section III.A above can be formulated foruse in accordance with any of the first, second or third aspect of thepresent invention and may, for example, be formulated in a way that issuitable for enteral, parenteral, topical, or pulmonary administration.

The compounds as defined in section III.A above can be combined with oneor more pharmaceutically acceptable carriers and/or excipients that areconsidered safe and effective and may be administered to an individualwithout causing undesirable biological side effects or unwantedinteractions.

The carrier can include all components present in the pharmaceuticalformulation other than the active ingredient or ingredients. Thecompounds are included in the formulation in an effective amount toachieve the effect of the first, second or third aspects of the presentinvention, for example in an amount that is effective to inhibit biofilmformation or reduce biofilm buildup. An effective amount of a compoundprovided to a subject may be an amount that is enough to provide therequired degree of reduction of microbial colonization. This may dependon the type of compound and/or the size of the animal.

In one embodiment an effective amount of the compound may be an amountthat is effective to deliver the compound to the site at which action isrequired in a concentration that ranges from 1 μm to 1 M, preferablygreater than 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180μM, 190 μM, 200 μM or more. A suitable concentration may be within therange of about 1 μm to about 1 mM, or about 30 μm to about 0.5 mM, orabout 60 μM to about 0.3 mM. These concentrations may particularly applyto the performance of the invention in the context of the second and/orthird aspects of the present invention.

In a further embodiment an effective amount of the compound may be 0.3to 32 mg/day/kg bodyweight of the subject such as a chicken. In anotherembodiment an effective concentration of the compound may be between0.001 to 1 mM for use in coatings or devices, or solutions.

The compounds can also be formulated for use as a disinfectant, forexample, in a hospital environment or for industrial application.

1. Parenteral Formulations

The compounds as defined in section III.A above for use in accordancewith any of the first, second or third aspect of the present inventionand may be formulated for parenteral administration.

Parenteral administration may include administration to a patientintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intravitreally, intratumorally,intramuscularly, subcutaneously, subconjunctivally, intravesicularly,intrapericardially, intraumbilically, by injection, and by infusion.

Parenteral formulations can be prepared as aqueous compositions usingtechniques known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions and dispersions of the active compounds as the free acid orbase or pharmacologically acceptable salts thereof can be prepared inwater or another solvent or dispersing medium suitably mixed with one ormore pharmaceutically acceptable excipients including, but not limitedto, surfactants, dispersants, emulsifiers, pH modifying agents,viscosity modifying agents, and combination thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface-active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium ionsof long chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® (triblockcopolymer of polyoxyethylene, followed by a block of polyoxypropylene,followed by a block of polyoxyethylene) 401, stearoylmonoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-β-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.It is to be noted that FeQ and some of the other compounds as defined inSection III.A of the application are acidic, and so advantageously areformulated with a buffer in order to achieve a suitable pH, particularlyin the context of preparing injectable formulation, includingformulations for intravenous injection.

Water-soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are well known in theart.

(a) Controlled Release Formulations

The parenteral formulations described herein comprising one or morecompounds as defined in section III.A above for use in accordance withany of the first, second or third aspect of the present invention may beformulated for controlled release including immediate release, delayedrelease, extended release, pulsatile release, and combinations thereof.

1. Nano- and Microparticles

For parenteral administration, the one or more compounds as defined insection III.A above for use in accordance with any of the first, secondor third aspect of the present invention, and optional one or moreadditional active agents, can be incorporated into microparticles,nanoparticles, or combinations thereof that provide controlled releaseof the compounds and/or one or more additional active agents. Inembodiments wherein the formulations contains two or more activecomponents, such as drugs, then they can be formulated for the same typeof controlled release (e.g., delayed, extended, immediate, or pulsatile)or they can be independently formulated for different types of release(e.g., immediate and delayed, immediate and extended, delayed andextended, delayed and pulsatile, etc.).

For example, the compounds and/or one or more additional active agentscan be incorporated into polymeric microparticles, which providecontrolled release of the active agent(s). Release of the active agent(s) is controlled by diffusion of the drug(s) out of the microparticlesand/or degradation of the polymeric particles by hydrolysis and/orenzymatic degradation. Suitable polymers include ethylcellulose andother natural or synthetic cellulose derivatives.

Polymers, which are slowly soluble and form a gel in an aqueousenvironment, such as hydroxypropyl methylcellulose or polyethyleneoxide, can also be suitable as materials for drug containingmicroparticles. Other polymers include, but are not limited to,polyanhydrides, poly(ester anhydrides), polyesters, such as polylactide(PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),polydioxanone, poly-3-hydroxybutyrate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof polycaprolactoneand copolymers thereof, polymers including, but not limited to, polymersof glycolic acid, lactic acid, 1,4-dioxanone, trimethylene carbonate,3-hydroxybutyric acid, 4-hydroxybutyrate, e-caprolactone, includingpolyglycolic acid, polylactic acid, polydioxanone, polycaprolactone,copolymers of glycolic and lactic acids, such as VICRYL® polymer, MAXON®and MONOCRYL® polymers, and including poly(lactide-co-caprolactones);poly(orthoesters); polyanhydrides; poly(phosphazenes);polyhydroxyalkanoates; synthetically or biologically preparedpolyesters; polycarbonates; tyrosine polycarbonates; polyamides(including synthetic and natural polyamides, polypeptides, andpoly(amino acids)); polyesteramides; poly(alkylene alkylates);polyethers (such as polyethylene glycol, PEG, and polyethylene oxide,PEO); polyvinyl pyrrolidones or PVP; polyurethanes; polyetheresters;polyacetals; polycyanoacrylates; poly(oxyethylene)/poly(oxypropylene)copolymers; polyacetals, polyketals; polyphosphates;(phosphorous-containing) polymers; polyphosphoesters; polyalkyleneoxalates; polyalkylene succinates; poly(maleic acids); silk (includingrecombinant silks and silk derivatives and analogs); chitin; chitosan;modified chitosan; biocompatible polysaccharides; hydrophilic or watersoluble polymers, such as polyethylene glycol, (PEG) or polyvinylpyrrolidone (PVP), with blocks of other biocompatible or biodegradablepolymers, for example, poly(lactide), poly(lactide-co-glycolide, orpolycaprolcatone and copolymers thereof, including random copolymers andblock copolymers thereof, and combinations thereof.

Alternatively, the active agent can be incorporated into microparticlesprepared from materials which are insoluble in aqueous solution orslowly soluble in aqueous solution, but are capable of degrading withinthe GI tract by means including enzymatic degradation, surfactant actionof bile acids, and/or mechanical erosion. As used herein, the term“slowly soluble in water” refers to materials that are not dissolved inwater within a period of 30 minutes. Preferred examples include fats,fatty substances, waxes, wax-like substances and mixtures thereof.Suitable fats and fatty substances include fatty alcohols (such aslauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids andderivatives, including but not limited to fatty acid esters, fatty acidglycerides (mono-, di- and tri-glycerides), and hydrogenated fats.Specific examples include, but are not limited to hydrogenated vegetableoil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenatedoils available under the trade name STEROTEX®, stearic acid, cocoabutter, and stearyl alcohol. Suitable waxes and wax-like materialsinclude natural or synthetic waxes, hydrocarbons, and normal waxes.Specific examples of waxes include beeswax, glycowax, castor wax,carnauba wax, paraffins and candelilla wax. As used herein, a wax-likematerial is defined as any material, which is normally solid at roomtemperature and has a melting point of from about 30 to 300° C.

In some cases, it may be desirable to alter the rate of waterpenetration into the microparticles. To this end, rate-controlling(wicking) agents can be formulated along with the fats or waxes listedabove. Examples of rate-controlling materials include certain starchderivatives (e.g., waxy maltodextrin and drum dried corn starch),cellulose derivatives (e.g., hydroxypropylmethyl-cellulose,hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose),alginic acid, lactose and talc. Additionally, a pharmaceuticallyacceptable surfactant (for example, lecithin) may be added to facilitatethe degradation of such microparticles.

Proteins, which are water insoluble, such as zein, can also be used asmaterials for the formation of active agent containing microparticles.Additionally, proteins, polysaccharides and combinations thereof, whichare water-soluble, can be formulated with drug into microparticles andsubsequently cross-linked to form an insoluble network. For example,cyclodextrins can be complexed with individual drug molecules andsubsequently cross-linked.

2. Method of Making Nano- and Microparticles

Encapsulation or incorporation of active agent, such as the one or morecompounds as defined in section III.A above for use in accordance withany of the first, second or third aspect of the present invention, intocarrier materials to produce drug-containing microparticles can beachieved through known pharmaceutical formulation techniques. In thecase of formulation in fats, waxes or wax-like materials, the carriermaterial is typically heated above its melting temperature and theactive agent is added to form a mixture comprising active agentparticles suspended in the carrier material, active agent dissolved inthe carrier material, or a mixture thereof. Microparticles can besubsequently formulated through several methods including, but notlimited to, the processes of congealing, extrusion, spray chilling oraqueous dispersion. In a preferred process, wax is heated above itsmelting temperature, active agent is added, and the molten wax-drugmixture is congealed under constant stirring as the mixture cools.Alternatively, the molten wax-drug mixture can be extruded andspheronized to form pellets or beads. These processes are known in theart.

For some carrier materials it may be desirable to use a solventevaporation technique to produce active agent-containing microparticles.In this case active agent and carrier material are co-dissolved in amutual solvent and microparticles can subsequently be produced byseveral techniques including, but not limited to, forming an emulsion inwater or other appropriate media, spray drying or by evaporating off thesolvent from the bulk solution and milling the resulting material.

In some embodiments, active agent in a particulate form is homogeneouslydispersed in a water-insoluble or slowly water soluble material. Tominimize the size of the active agent particles within the composition,the active agent powder itself may be milled to generate fine particlesprior to formulation. The process of jet milling, known in thepharmaceutical art, can be used for this purpose. In some embodimentsactive agent in a particulate form is homogeneously dispersed in a waxor wax like substance by heating the wax or wax like substance above itsmelting point and adding the active agent particles while stirring themixture. In this case a pharmaceutically acceptable surfactant may beadded to the mixture to facilitate the dispersion of the active agentparticles.

The particles can also be coated with one or more modified releasecoatings. Solid esters of fatty acids, which are hydrolyzed by lipases,can be spray coated onto microparticles or active agent particles. Zeinis an example of a naturally water-insoluble protein. It can be coatedonto active agent containing microparticles or active agent particles byspray coating or by wet granulation techniques. In addition to naturallywater-insoluble materials, some substrates of digestive enzymes can betreated with cross-linking procedures, resulting in the formation ofnon-soluble networks. Many methods of cross-linking proteins, initiatedby both chemical and physical means, have been reported. One of the mostcommon methods to obtain cross-linking is the use of chemicalcross-linking agents. Examples of chemical cross-linking agents includealdehydes (gluteraldehyde and formaldehyde), epoxy compounds,carbodiimides, and genipin. In addition to these cross-linking agents,oxidized and native sugars have been used to cross-link gelatin.Cross-linking can also be accomplished using enzymatic means; forexample, transglutaminase has been approved as a GRAS substance forcross-linking seafood products. Finally, cross-linking can be initiatedby physical means such as thermal treatment, UV irradiation and gammairradiation.

To produce a coating layer of cross-linked protein surrounding activeagent containing microparticles or active agent particles, awater-soluble protein can be spray coated onto the microparticles andsubsequently cross-linked by the one of the methods described above.Alternatively, active agent-containing microparticles can bemicroencapsulated within protein by coacervation-phase separation (forexample, by the addition of salts) and subsequently cross-linked. Somesuitable proteins for this purpose include gelatin, albumin, casein, andgluten.

Polysaccharides can also be cross-linked to form a water-insolublenetwork. For many polysaccharides, this can be accomplished by reactionwith calcium salts or multivalent cations, which cross-link the mainpolymer chains. Pectin, alginate, dextran, amylose and guar gum aresubject to cross-linking in the presence of multivalent cations.Complexes between oppositely charged polysaccharides can also be formed;pectin and chitosan, for example, can be complexed via electrostaticinteractions.

(b) Injectable/Implantable Formulations

The one or more compounds as defined in section III.A above for use inaccordance with any of the first, second or third aspect of the presentinvention can be incorporated into injectable/implantable solid orsemi-solid implants, such as polymeric implants. In one embodiment, thecompounds are incorporated into a polymer that is a liquid or paste atroom temperature, but upon contact with aqueous medium, such asphysiological fluids, exhibits an increase in viscosity to form asemi-solid or solid material. Exemplary polymers include, but are notlimited to, hydroxyalkanoic acid polyesters derived from thecopolymerization of at least one unsaturated hydroxy fatty acidcopolymerized with hydroxyalkanoic acids. The polymer can be melted,mixed with the active substance and cast or injection molded into adevice. Such melt fabrication require polymers having a melting pointthat is below the temperature at which the substance to be delivered andpolymer degrade or become reactive. The device can also be prepared bysolvent casting where the polymer is dissolved in a solvent and the drugdissolved or dispersed in the polymer solution and the solvent is thenevaporated. Solvent processes require that the polymer be soluble inorganic solvents. Another method is compression molding of a mixedpowder of the polymer and the drug or polymer particles loaded with theactive agent.

Alternatively, the compounds can be incorporated into a polymer matrixand molded, compressed, or extruded into a device that is a solid atroom temperature. For example, the compounds can be incorporated into abiodegradable polymer, such as polyanhydrides, polyhydroalkanoic acids(PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides,polyorthoesters, polyphosphazenes, proteins and polysaccharides such ascollagen, hyaluronic acid, albumin and gelatin, and combinations thereofand compressed into solid device, such as disks, or extruded into adevice, such as rods. Further alternative polymers for use in thiscontext include polymers include, but are not limited to, polymers ofglycolic acid, lactic acid, 1,4-dioxanone, trimethylene carbonate,3-hydroxybutyric acid, 4-hydroxybutyrate, e-caprolactone, includingpolyglycolic acid, polylactic acid, polydioxanone, polycaprolactone,copolymers of glycolic and lactic acids, such as VICRYL® polymer, MAXON®and MONOCRYL® polymers, and including poly(lactide-co-caprolactones);poly(orthoesters); polyanhydrides; poly(phosphazenes);polyhydroxyalkanoates; synthetically or biologically preparedpolyesters; polycarbonates; tyrosine polycarbonates; polyamides(including synthetic and natural polyamides, polypeptides, andpoly(amino acids)); polyesteramides; poly(alkylene alkylates);polyethers (such as polyethylene glycol, PEG, and polyethylene oxide,PEO); polyvinyl pyrrolidones or PVP; polyurethanes; polyetheresters;polyacetals; polycyanoacrylates; poly(oxyethylene)/poly(oxypropylene)copolymers; polyacetals, polyketals; polyphosphates;(phosphorous-containing) polymers; polyphosphoesters; polyalkyleneoxalates; polyalkylene succinates; poly(maleic acids); silk (includingrecombinant silks and silk derivatives and analogs); chitin; chitosan;modified chitosan; biocompatible polysaccharides; hydrophilic or watersoluble polymers, such as polyethylene glycol, (PEG) or polyvinylpyrrolidone (PVP), with blocks of other biocompatible or biodegradablepolymers, for example, poly(lactide), poly(lactide-co-glycolide, orpolycaprolcatone and copolymers thereof, including random copolymers andblock copolymers thereof.

The release of the one or more compounds from the implant can be variedby selection of the polymer, the molecular weight of the polymer, and/ormodification of the polymer to increase degradation, such as theformation of pores and/or incorporation of hydrolyzable linkages.Methods for modifying the properties of biodegradable polymers to varythe release profile of the compounds from the implant are well known inthe art.

2. Enteral Formulations

The compounds as defined in section III.A above for use in accordancewith any of the first, second or third aspect of the present inventionand may be formulated for enteral administration.

Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can be prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art.

Formulations may be prepared using a pharmaceutically acceptablecarrier. As generally used herein “carrier” includes, but is not limitedto, diluents, preservatives, binders, lubricants, disintegrators,swelling agents, fillers, stabilizers, and combinations thereof.

Carrier also includes all components of the coating composition, whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

“Diluents”, also referred to as “fillers,” are typically necessary toincrease the bulk of a solid dosage form so that a practical size isprovided for compression of tablets or formation of beads and granules.Suitable diluents include, but are not limited to, dicalcium phosphatedihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,cellulose, microcrystalline cellulose, kaolin, sodium chloride, drystarch, hydrolyzed starches, pregelatinized starch, silicone dioxide,titanium oxide, magnesium aluminum silicate and powdered sugar.

“Binders” are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

“Lubricants” are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

“Disintegrants” are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (POLYPLASDONE® XL from GAF ChemicalCorp).

“Stabilizers” are used to inhibit or retard drug decompositionreactions, which include, by way of example, oxidative reactions.Suitable stabilizers include, but are not limited to, antioxidants,butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters;Vitamin E, tocopherol and its salts; sulfites such as sodiummetabisulphite; cysteine and its derivatives; citric acid; propylgallate, and butylated hydroxyanisole (BHA).

(a) Controlled Release Enteral Formulations

Oral dosage forms, such as capsules, tablets, solutions, andsuspensions, can be formulated for controlled release, for example, forthe controlled release of the one or more compounds as defined insection III.A above for use in accordance with any of the first, secondor third aspect of the present invention. For example, the one or morecompounds and optional one or more additional active agents can beformulated into nanoparticles, microparticles, and combinations thereof,and encapsulated in a soft or hard gelatin or non-gelatin capsule ordispersed in a dispersing medium to form an oral suspension or syrup.The particles can be formed of the active agent and a controlled releasepolymer or matrix. Alternatively, the active agent particles can becoated with one or more controlled release coatings prior toincorporation in to the finished dosage form.

In another embodiment, the one or more compounds and optional one ormore additional active agents are dispersed in a matrix material, whichgels or emulsifies upon contact with an aqueous medium, such asphysiological fluids. In the case of gels, the matrix swells entrappingthe active agents, which are released slowly over time by diffusionand/or degradation of the matrix material. Such matrices can beformulated as tablets or as fill materials for hard and soft capsules.

In still another embodiment, the one or more compounds, and optional oneor more additional active agents are formulated into a sold oral dosageform, such as a tablet or capsule, and the solid dosage form is coatedwith one or more controlled release coatings, such as a delayed releasecoatings or extended release coatings. The coating or coatings may alsocontain the compounds and/or additional active agents.

(1) Extended Release Dosage Forms

The extended release formulations are generally prepared as diffusion orosmotic systems, which are known in the art. A diffusion systemtypically consists of two types of devices, a reservoir and a matrix,and is well known and described in the art. The matrix devices aregenerally prepared by compressing the drug with a slowly dissolvingpolymer carrier into a tablet form. The three major types of materialsused in the preparation of matrix devices are insoluble plastics,hydrophilic polymers, and fatty compounds. Plastic matrices include, butare not limited to, methyl acrylate-methyl methacrylate, polyvinylchloride, and polyethylene. Hydrophilic polymers include, but are notlimited to, cellulosic polymers such as methyl and ethyl cellulose,hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCARBOPOL® 934 (cross-linked polyacrylate polymer), polyethylene oxidesand mixtures thereof. Fatty compounds include, but are not limited to,various waxes such as carnauba wax and glyceryl tristearate and wax-typesubstances including hydrogenated castor oil or hydrogenated vegetableoil, or mixtures thereof.

In certain preferred embodiments, the plastic material is apharmaceutically acceptable acrylic polymer, including but not limitedto, acrylic acid and methacrylic acid copolymers, methyl methacrylate,methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid),poly(methacrylic acid), methacrylic acid alkylamine copolymerpoly(methyl methacrylate), poly(methacrylic acid)(anhydride),polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), andglycidyl methacrylate copolymers.

In certain preferred embodiments, the acrylic polymer is comprised ofone or more ammonio methacrylate copolymers. Ammonio methacrylatecopolymers are well known in the art, and are described in NF XVII asfully polymerized copolymers of acrylic and methacrylic acid esters witha low content of quaternary ammonium groups.

In one preferred embodiment, the acrylic polymer is an acrylic resinlacquer such as that which is commercially available from Rohm Pharmaunder the tradename EUDRAGIT®. In further preferred embodiments, theacrylic polymer comprises a mixture of two acrylic resin lacquerscommercially available from Rohm Pharma under the trade names EUDRAGIT®RL30D and EUDRAGIT® RS30D, respectively. EUDRAGIT® RL30D and EUDRAGIT®RS30D are copolymers of acrylic and methacrylic esters with a lowcontent of quaternary ammonium groups, the molar ratio of ammoniumgroups to the remaining neutral (meth)acrylic esters being 1:20 inEUDRAGIT® RL30D and 1:40 in EUDRAGIT® RS30D. The mean molecular weightis about 150,000. EUDRAGIT® S-100 and EUDRAGIT® L-100 are alsopreferred. The code designations RL (high permeability) and RS (lowpermeability) refer to the permeability properties of these agents.EUDRAGIT® RL/RS mixtures are insoluble in water and in digestive fluids.However, multiparticulate systems formed to include the same areswellable and permeable in aqueous solutions and digestive fluids.

The polymers described above such as EUDRAGIT® RL/RS may be mixedtogether in any desired ratio in order to ultimately obtain asustained-release formulation having a desirable dissolution profile.Desirable sustained-release multiparticulate systems may be obtained,for instance, from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGITT® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS. One skilled in the artwill recognize that other acrylic polymers may also be used, such as,for example, EUDRAGIT® L.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets and capsules containing tablets, beads, or granules. Animmediate release portion can be added to the extended release system bymeans of either applying an immediate release layer on top of theextended release core using a coating or compression process or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In the congealingmethod, the drug is mixed with a wax material and either spray-congealedor congealed and screened and processed.

(2) Delayed Release Dosage Forms

Delayed release formulations can be created by coating a solid dosageform with a polymer film, which is insoluble in the acidic environmentof the stomach, and soluble in the neutral environment of the smallintestine.

The delayed release dosage units can be prepared, for example, bycoating an active agent or an active agent-containing composition with aselected coating material. The active agent-containing composition maybe, e.g., a tablet for incorporation into a capsule, a tablet for use asan inner core in a “coated core” dosage form, or a plurality of activeagent-containing beads, particles or granules, for incorporation intoeither a tablet or capsule. Preferred coating materials includebioerodible, gradually hydrolyzable, gradually water-soluble, and/orenzymatically degradable polymers, and may be conventional “enteric”polymers. Enteric polymers, as will be appreciated by those skilled inthe art, become soluble in the higher pH environment of the lowergastrointestinal tract or slowly erode as the dosage form passes throughthe gastrointestinal tract, while enzymatically degradable polymers aredegraded by bacterial enzymes present in the lower gastrointestinaltract, particularly in the colon. Suitable coating materials foreffecting delayed release include, but are not limited to, cellulosicpolymers such as hydroxypropyl cellulose, hydroxyethyl cellulose,hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropylmethyl cellulose acetate succinate, hydroxypropylmethyl cellulosephthalate, methylcellulose, ethyl cellulose, cellulose acetate,cellulose acetate phthalate, cellulose acetate trimellitate andcarboxymethylcellulose sodium; acrylic acid polymers and copolymers,preferably formed from acrylic acid, methacrylic acid, methyl acrylate,ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and othermethacrylic resins that are commercially available under the tradenameEUDRAGIT® (Rohm Pharma; Westerstadt, Germany), including EUDRAGIT®L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT® L-100(soluble at pH 6.0 and above), EUDRAGIT® S (soluble at pH 7.0 and above,as a result of a higher degree of esterification), and EUDRAGITS® NE, RLand RS (water-insoluble polymers having different degrees ofpermeability and expandability); vinyl polymers and copolymers such aspolyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate,vinylacetate crotonic acid copolymer, and ethylene-vinyl acetatecopolymer, enzymatically degradable polymers such as azo polymers,pectin, chitosan, amylose and guar gum; zein and shellac. Combinationsof different coating materials may also be used. Multi-layer coatingsusing different polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 3 wt. % to 50 wt. %”, or 10 wt % to50 wt. %, relative to the dry weight of the polymer. Examples of typicalplasticizers include polyethylene glycol, propylene glycol, triacetin,dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutylsebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate,castor oil and acetylated monoglycerides. A stabilizing agent ispreferably used to stabilize particles in the dispersion. Typicalstabilizing agents are nonionic emulsifiers such as sorbitan esters,polysorbates and polyvinylpyrrolidone. Glidants are recommended toreduce sticking effects during film formation and drying, and willgenerally represent approximately 25 wt. % to 100 wt. % of the polymerweight in the coating solution. One effective glidant is talc. Otherglidants such as magnesium stearate and glycerol monostearates may alsobe used. Pigments such as titanium dioxide may also be used. Smallquantities of an anti-foaming agent, such as a silicone (e.g.,simethicone), may also be added to the coating composition.

3. Topical Formulations

The compounds as defined in section III.A above for use in accordancewith any of the first, second or third aspect of the present inventionand may be formulated for topical administration.

The formulations may contain the one or more compounds discussed above,alone or in combination, in an effective amount to prevent or inhibitbiofilm formation on a surface, or reduce the amount of biofilm on asurface being treated. 1000 colony forming units (cfu) of Campylobacterare enough to infect a human and cause disease in a human.

Therefore, in one embodiment, an effective amount of the one or morecompounds as defined in section III.A of this application is, or are,enough of the compound(s), alone, or in combination with othercompounds, to reduce the number of cfu of Campylobacter or othermicroorganism of interest on the surface being treated to a number thatis unlikely to, or which will not, cause infection in humans.

Suitable dosage forms for topical administration include creams,ointments, salves, sprays, gels, lotions, irrigants, and emulsions.

“Buffers” are used to control pH of a composition. Preferably, thebuffers buffer the composition from a pH of about 4 to a pH of about7.5, more preferably from a pH of about 4 to a pH of about 7, and mostpreferably from a pH of about 5 to a pH of about 7. In a preferredembodiment, the buffer is triethanolamine.

“Emollients” are an externally applied agent that softens or soothesskin and are generally known in the art and listed in compendia, such asthe “Handbook of Pharmaceutical Excipients”, 4^(th) Ed., PharmaceuticalPress, 2003. These include, without limitation, almond oil, castor oil,ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esterswax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycolpalmitostearate, glycerin, glycerin monostearate, glyceryl monooleate,isopropyl myristate, isopropyl palmitate, lanolin, lecithin, lightmineral oil, medium-chain triglycerides, mineral oil and lanolinalcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil,starch, stearyl alcohol, sunflower oil, xylitol and combinationsthereof. In one embodiment, the emollients are ethylhexylstearate andethylhexyl palmitate.

“Emulsifiers” are surface active substances which promote the suspensionof one liquid in another and promote the formation of a stable mixture,or emulsion, of oil and water. Common emulsifiers are: metallic soaps,certain animal and vegetable oils, and various polar compounds. Suitableemulsifiers include acacia, anionic emulsifying wax, calcium stearate,carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol,diethanolamine, ethylene glycol palmitostearate, glycerin monostearate,glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin,hydrous, lanolin alcohols, lecithin, medium-chain triglycerides,methylcellulose, mineral oil and lanolin alcohols, monobasic sodiumphosphate, monoethanolamine, nonionic emulsifying wax, oleic acid,poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylenecastor oil derivatives, polyoxyethylene sorbitan fatty acid esters,polyoxyethylene stearates, propylene glycol alginate, self-emulsifyingglyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate,sorbitan esters, stearic acid, sunflower oil, tragacanth,triethanolamine, xanthan gum and combinations thereof. In oneembodiment, the emulsifier is glycerol stearate.

“Penetration enhancers” are known in the art and include, but are notlimited to, fatty alcohols, fatty acid esters, fatty acids, fattyalcohol ethers, amino acids, phospholipids, lecithins, cholate salts,enzymes, amines and amides, complexing agents (liposomes, cyclodextrins,modified celluloses, and diimides), macrocyclics, such as macrocyliclactones, ketones, and anhydrides and cyclic ureas, surfactants,N-methyl pyrrolidones and derivatives thereof, DMSO and relatedcompounds, ionic compounds, azone and related compounds, and solvents,such as alcohols, ketones, amides, polyols (e.g., glycols). Examples ofthese classes are known in the art.

“Preservatives” can be used to prevent the growth of fungi andmicroorganisms. Suitable antifungal and antimicrobial agents include,but are not limited to, benzoic acid, butylparaben, ethyl paraben,methyl paraben, propylparaben, sodium benzoate, sodium propionate,benzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,and thimerosal.

“Surfactants” are surface-active agents that lower surface tension andthereby increase the emulsifying, foaming, dispersing, spreading andwetting properties of a product. Suitable non-ionic surfactants includeemulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers,polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters,benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate,poloxamer, povidone and combinations thereof. In one embodiment, thenon-ionic surfactant is stearyl alcohol.

(a) Emulsions

An emulsion is a preparation of one liquid distributed in small globulesthroughout the body of a second liquid. In particular embodiments, thenon-miscible components of the emulsion include a lipophilic componentand an aqueous component. The dispersed liquid is the discontinuousphase, and the dispersion medium is the continuous phase. When oil isthe dispersed liquid and an aqueous solution is the continuous phase, itis known as an oil-in-water emulsion, whereas when water or aqueoussolution is the dispersed phase and oil or oleaginous substance is thecontinuous phase, it is known as a water-in-oil emulsion. Either or bothof the oil phase and the aqueous phase may contain one or moresurfactants, emulsifiers, emulsion stabilizers, buffers, and otherexcipients. Preferred excipients include surfactants, especiallynon-ionic surfactants; emulsifying agents, especially emulsifying waxes;and liquid non-volatile non-aqueous materials, particularly glycols suchas propylene glycol. The oil phase may contain other oilypharmaceutically approved excipients. For example, materials such ashydroxylated castor oil or sesame oil may be used in the oil phase assurfactants or emulsifiers.

The oil phase may consist at least in part of a propellant, such as anHFA propellant. Either or both of the oil phase and the aqueous phasemay contain one or more surfactants, emulsifiers, emulsion stabilizers,buffers, and other excipients. Preferred excipients include surfactants,especially non-ionic surfactants; emulsifying agents, especiallyemulsifying waxes; and liquid non-volatile non-aqueous materials,particularly glycols such as propylene glycol. The oil phase may containother oily pharmaceutically approved excipients. For example, materialssuch as hydroxylated castor oil or sesame oil may be used in the oilphase as surfactants or emulsifiers.

A sub-set of emulsions are the self-emulsifying systems. These deliverysystems are typically capsules (hard shell or soft shell) comprised ofthe compound dispersed or dissolved in a mixture of surfactant(s) andlipophilic liquids such as oils or other water immiscible liquids. Whenthe capsule is exposed to an aqueous environment and the outer gelatinshell dissolves, contact between the aqueous medium and the capsulecontents instantly generates very small emulsion droplets. Thesetypically are in the size range of micelles or nanoparticles. No mixingforce is required to generate the emulsion as is typically the case inemulsion formulation processes.

(b) Lotions

A lotion can contain finely powdered substances that are insoluble inthe dispersion medium through the use of suspending agents anddispersing agents. Alternatively, lotions can have as the dispersedphase liquid substances that are immiscible with the vehicle and areusually dispersed by means of emulsifying agents or other suitablestabilizers. In one embodiment, the lotion is in the form of an emulsionhaving a viscosity of between 100 and 1000 centistokes. The fluidity oflotions permits rapid and uniform application over a wide surface area.Lotions are typically intended to dry on the skin leaving a thin coat oftheir medicinal components on the skin's surface.

(c) Creams

Creams may contain emulsifying agents and/or other stabilizing agents.In one embodiment, the formulation is in the form of a cream having aviscosity of greater than 1000 centistokes, typically in the range of20,000-50,000 centistokes. Creams are often time preferred overointments, as they are generally easier to spread and easier to remove.

The difference between a cream and a lotion is the viscosity, which isdependent on the amount/use of various oils and the percentage of waterused to prepare the formulations. Creams are typically thicker thanlotions, may have various uses and often one uses more variedoils/butters, depending upon the desired effect upon the skin. In acream formulation, the water-base percentage is about 60-75% and theoil-base is about 20-30% of the total, with the other percentages beingthe emulsifier agent, preservatives and additives for a total of 100%.

(d) Ointments

Examples of suitable ointment bases include hydrocarbon bases (e.g.,petrolatum, white petrolatum, yellow ointment, and mineral oil);absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin,and cold cream); water-removable bases (e.g., hydrophilic ointment), andwater-soluble bases (e.g., polyethylene glycol ointments). Pastestypically differ from ointments in that they contain a larger percentageof solids. Pastes are typically more absorptive and less greasy thanointments prepared with the same components.

(e) Gels

Gels are semisolid systems containing dispersions of small or largemolecules in a liquid vehicle that is rendered semisolid by the actionof a thickening agent or polymeric material dissolved or suspended inthe liquid vehicle. The liquid may include a lipophilic component, anaqueous component or both. Some emulsions may be gels or otherwiseinclude a gel component. Some gels, however, are not emulsions becausethey do not contain a homogenized blend of immiscible components.Suitable gelling agents include, but are not limited to, modifiedcelluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose;Carbopol homopolymers and copolymers; and combinations thereof. Suitablesolvents in the liquid vehicle include, but are not limited to, diglycolmonoethyl ether; alkylene glycols, such as propylene glycol; dimethylisosorbide; alcohols, such as isopropyl alcohol and ethanol. Thesolvents are typically selected for their ability to dissolve thecompound. Other additives, which improve the skin feel and/or emolliencyof the formulation, may also be incorporated. Examples of such additivesinclude, but are not limited to, isopropyl myristate, ethyl acetate,C₁₂-C₁₅ alkyl benzoates, mineral oil, squalane, cyclomethicone,capric/caprylic triglycerides, and combinations thereof.

(f) Foams

Foams consist of an emulsion in combination with a gaseous propellant.The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs).Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures andadmixtures of these and other HFAs that are currently approved or maybecome approved for medical use are suitable. The propellants preferablyare not hydrocarbon propellant gases, which can produce flammable orexplosive vapors during spraying. Furthermore, the compositionspreferably contain no volatile alcohols, which can produce flammable orexplosive vapors during use.

4. Disinfecting and Cleaning Formulations

The compounds as defined in section III.A above for use in accordancewith any of the first, second or third aspect of the present inventionmay be formulated into cleaning formulations.

The cleaning formulations include formulations that are highlyefficacious for household cleaning applications (e.g., hard surfaceslike floors, countertops, tubs, tile, dishes and softer cloth materialslike clothing, sponges, paper towels, etc.), personal care applications(e.g. lotions, shower gels, soaps, shampoos, sprays, wipes, toothpaste,acne treatments, skin cleansers, mouthwash, wound irrigation solutions,towelettes, contact lenses and lens cases) and industrial and hospitalapplications (e.g., antifouling coatings, and disinfection ofinstruments, medical devices, gloves, filters, membranes, tubing,drains, pipes including gas pipes, oil pipes, drilling pipes, frackingpipes, sewage pipes, drainage pipes, hoses, animal carcasses, fishtanks, showers, children's toys, boat hulls, and cooling towers). Theseformulations are efficacious for cleaning surfaces which are infected orcontaminated with biofilm or for preventing the formation of biofilm onthese surfaces.

The compounds can be formulated into a solution in a suitable solventfor administration in a spray bottle, the compounds can be formulated asan aerosol, as a foam, suitable for spraying onto surfaces, or, they canbe imbibed into a cloth or other item suitable for wiping down a surfaceto be disinfected. Methods for making formulations for use as adisinfectant in the forms are known in the art.

One embodiment provides the compounds or a derivative thereof in acomposition containing a pH dye indicator and an alkaline substance. ThepH indicator dye indicates what surface has been disinfected and ensuresthat a sufficient time has passed to disinfect the surface. See forexample, U.S. Publication No. 20140057987, which is incorporated byreference in its entirety.

Cleaning formulations can include the compounds and an acceptablecarrier. The carrier can be in a wide variety of forms. For example, thecarrier may be an aqueous-based solution or cleanser, an alcohol-basedsolution or gel or an emulsion carrier, including, but not limited to,oil-in-water, water-in-oil, water-in-oil-in-water, andoil-in-water-in-silicone emulsions. The carrier solution containing thecompound(s) can be applied directly to the surface to be treated ordelivered via a suitable substrate.

The cleaning formulations can be formulated for use on the skin. Inthese embodiments the compounds can be formulate in a dermatologicallyacceptable carrier. The dermatologically acceptable carriers can alsobe, for example, formulated as alcohol or water based hand cleansers,toilet bars, liquid soaps, shampoos, bath gels, hair conditioners, hairtonics, pastes, or mousses.

Cleaning formulations can contain one or more surfactants. Thesurfactant is suitably selected from anionic, nonionic, zwitterionic,amphoteric and ampholytic surfactants, as well as mixtures of thesesurfactants. Such surfactants are well known to those skilled in thedetergency art. Non limiting examples of possible surfactants includeisoceteth-20, sodium methyl cocoyl taurate, sodium methyl oleoyltaurate, and sodium lauryl sulfate. Examples of a broad variety ofadditional surfactants are described in McCutcheon's Detergents andEmulsifiers. North American Edition (1986), published by AlluredPublishing Corporation. The cleansing formulations can optionallycontain, at their art-established levels, other materials which areconventionally used in cleansing formulations.

Additional carriers suitable for the cleaning formulations may includevarious substrate-based products. In such instances, the presentformulations may be impregnated into or onto the substrate products andmay be allowed to remain wet or may be subjected to a drying process.For instance, suitable carriers include, but are not limited to, dry andwet wipes suitable for personal care and household use (e.g., nonwovenbaby wipes, household cleaning wipes, surgical preparation wipes, etc.);diapers; infant changing pads; dental floss; personal care and householdcare sponges or woven cloths (e.g., washcloths, towels, etc.);tissue-type products (e.g. facial tissue, paper towels, etc.); anddisposable garments (e.g., gloves, smocks, surgical masks, infant bibs,socks, shoe inserts, etc.). Cleaning formulations can be incorporatedinto various household care products including, but not limited to, hardsurface cleaners (e.g., disinfectant sprays, liquids, or powders); dishor laundry detergents (liquid or solid), floor waxes, glass cleaners,etc.

Exemplary carriers can include aqueous solutions, e.g. having from about0% to about 98.8%, by weight of the composition, of water. Additionally,carriers may contain an aqueous alcohol solution. The amount of alcoholpresent in the alcohol solution will vary depending on the type ofproduct in which the composition is incorporated, i.e. say a wipe wherethe preferred amount of alcohol present would be from about 0% to about25% whereas a hand sanitizer preferably contains from about 60% to about95%, of alcohol. Therefore, suitable dermatologically acceptable alcoholsolutions or gels may contain from about 0% to about 95%, by weight ofthe composition, of an alcohol.

Alcohols suitable for inclusion in the alcohol solutions of the carrierinclude, but are not limited to, monohydric alcohols, dihydric alcohols,and combinations thereof. More preferred alcohols are selected from thegroup consisting of monohydric linear or branched C2-C18 alcohols. Themost preferred alcohols are selected from the group consisting ofethanol, isopropanol, n-propanol, butanol, and combinations thereof. Thecleaning formulations which contain an alcohol solution may be anhydrousor water containing.

Thickeners can be added to the water or alcohol based to form a gel.Examples of suitable thickeners include, but are not limited to,naturally-occurring polymeric materials such as sodium alginate, xanthangum, quince seed extract, tragacanth gum, starch and the like,semi-synthetic polymeric materials such as cellulose ethers (e.g.hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose,hydroxy propylmethyl cellulose), polyvinylpyrrolidone, polyvinylalcohol,guar gum, hydroxypropyl guar gum, soluble starch, cationic celluloses,cationic guars and the like and synthetic polymeric materials such ascarboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl alcohol,polyacrylic acid polymers, polymethacrylic acid polymers, polyvinylacetate polymers, polyvinyl chloride polymers, and polyvinylidenechloride polymers. Inorganic thickeners may also be used such asaluminum silicates, such as, for example, bentonites, or a mixture ofpolyethylene glycol and polyethylene glycol stearate or distearate.

The cleaning formulations can contain, in addition to the compoundsdescribed above, one or more antimicrobial or antifungal agents. Suchagents are capable of destroying microbes, preventing the development ofmicrobes or preventing the pathogenic action of microbes. Examples ofadditional antimicrobial and antifungal agents include β-lactam drugs,quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin,amikacin, 2,4,4′-trichloro-2′-hydroxy diphenyl ether (TRICLOSAN®),phenoxyethanol, phenoxy propanol, phenoxyisopropanol, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isethionate, metronidazole,pentamidine, gentamicin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmicin, paromomycin,streptomycin, tobramycin, miconazole, tetracycline hydrochloride,erythromycin, zinc erythromycin, erythromycin estolate, erythromycinstearate, amikacin sulfate, doxycycline hydrochloride, capreomycinsulfate, chlorhexidine gluconate, chlorhexidine hydrochloride,chlortetracycline hydrochloride, oxytetracycline hydrochloride,clindamycin hydrochloride, ethambutol hydrochloride, metronidazolehydrochloride, pentamidine hydrochloride, gentamicin sulfate, kanamycinsulfate, lineomycin hydrochloride, methacycline hydrochloride,methenamine hippurate, methenamine mandelate, minocycline hydrochloride,neomycin sulfate, netilmicin sulfate, paromomycin sulfate, streptomycinsulfate, tobramycin sulfate, miconazole hydrochloride, ketaconazole,amanfadine hydrochloride, amanfadine sulfate, octopirox, parachlorometaxylenol, nystatin, tolnaftate, pyrithiones (especially zinc pyrithionewhich is also known as ZPT), dimethyldimethylol hydantoin (GLYDANT®),methylchloroisothiazolinone/methylisothiazolinone (KATHON CG®), sodiumsulfite, sodium bisulfite, imidazolidinyl urea (Germall 115®),diazolidinyl urea (GERMAILL II®), benzyl alcohol,2-bromo-2-nitropropane-1,3-diol (BRONOPOL®), formalin (formaldehyde),iodopropenyl butylcarbamate (POLYPHASE P100®), chloroacetamide,methanamine, methyldibromonitrile glutaronitrile(1,2-Dibromo-2,4-dicyanobutane or TEKTAMER®), glutaraldehyde,5-bromo-5-nitro-1,3-dioxane (BRONIDOX®), phenethyl alcohol,o-phenylphenol/sodium o-phenylphenol, sodium hydroxymethylglycinate(SUTTOCIDE A®), polymethoxy bicyclic oxazolidine (NUOSEPt C®),dimethoxane, thimersal dichlorobenzyl alcohol, captan, chloφphenenesin,dichlorophene, chlorbutanol, glyceryl laurate, halogenated diphenylethers like 2,4,4′-trichloro-2′-hydroxy-diphenyl ether (TRICLOSAN® orTCS), 2,2′-dihydroxy-5,5′-dibromo-diphenyl ether, phenolic compoundslike phenol, 2-methyl phenol, 3-methyl phenol, 4-methyl phenol, 4-ethylphenol, 2,4-dimethyl phenol, 2,5-dimethyl pPhenol, 3,4-dimethyl phenol,2,6-dimethyl phenol, 4-n-propyl phenol, 4-n-butyl phenol, 4-n-amylphenol, 4-tert-amyl phenol, 4-n-hexyl phenol, 4-n-heptyl phenol, mono-and poly-alkyl and aromatic halophenols such as p-chlorophenol, methylp-chlorophenol, ethyl p-chlorophenol, n-propyl p-chlorophenol, n-butylp-chlorophenol, n-amyl p-chlorophenol, sec-amyl p-chlorophenol, n-hexylp-chlorophenol, cyclohexyl p-chlorophenol, n-heptyl p-chlorophenol,n-octyl p-chlorophenol, o-chlorophenol, methyl o-chlorophenol, ethylo-chlorophenol, n-propyl o-chlorophenol, n-butyl o-chlorophenol, n-amylo-chlorophenol, tert-amyl o-chlorophenol, n-hexyl o-chlorophenol,n-heptyl o-chlorophenol, o-benzyl p-chlorophenol, o-benzyl-m-methylp-chlorophenol, o-benzyl-m, m-dimethyl p-chlorophenol, o-phenylethylp-chlorophenol, o-phenylethyl-m-methyl p-chlorophenol, 3-methylp-chlorophenol, 3,5-dimethyl p-chlorophenol, 6-ethy 1-3-methylp-chlorophenol, 6-n-propyl-3-methyl p-chlorophenol,6-iso-propyl-3-methyl p-chlorophenol, 2-ethyl-3,5-dimethylp-chlorophenol, 6-sec-butyl-3-methyl p-chlorophenol,2-iso-propyl-3,5-dimethyl p-chlorophenol, 6-diethylmethyl-3-methylp-chlorophenol, 6-iso-propyl-2-ethyl-3-methyl p-chlorophenol,2-sec-amyl-3,5-dimethyl p-chlorophenol, 2-diethylmethyl-3,5-dimethylp-chlorophenol, 6-sec-octyl-3-methyl p-chlorophenol, p-chloro-m-cresol,p-bromophenol, methyl p-bromophenol, ethyl p-bromophenol, n-propylp-bromophenol, n-butyl p-bromophenol, n-amyl p-bromophenol, sec-amylp-bromophenol, n-hexyl p-bromophenol, cyclohexyl p-bromophenol,o-bromophenol, tert-amyl o-bromophenol, n-hexyl o-bromophenol,n-propyl-m,m-dimethyl o-bromophenol, 2-phenyl phenol, 4-chloro-2-methylphenol, 4-chloro-3-methyl phenol, 4-chloro-3,5-dimethyl phenol,2,4-dichloro-3,5-dimethylphenol, 3,4,5,6-terabromo-2-methylphenol,5-methyl-2-pentylphenol, 4-isopropyl-3-methylphenol,para-chloro-meta-xylenol (PCMX), chlorothymol,5-chloro-2-hydroxydiphenylmethane, resorcinol and its derivativesincluding methyl resorcinol, ethyl resorcinol, n-propyl resorcinol,n-butyl resorcinol, n-amyl resorcinol, n-hexyl resorcinol, n-heptylresorcinol, n-octyl resorcinol, n-nonyl resorcinol, phenyl resorcinol,benzyl resorcinol, phenylethyl resorcinol, phenylpropyl resorcinol,p-chlorobenzyl resorcinol, 5-chloro 2,4-dihydroxydiphenyl methane,4′-chloro 2,4-dihydroxydiphenyl methane, 5-bromo 2,4-dihydroxydiphenylmethane, and 4′-bromo 2,4-dihydroxydiphenyl methane, bisphenoliccompounds like 2,2′-methylene bis(4-chlorophenol), 2,2′-methylenebis(3,4,6-trichlorophenol), 2,2′-methylene bis(4-chloro-6-bromophenol),bis(2-hydroxy-3,5-dichlorophenyl) sulphide, andbis(2-hydroxy-5-chlorobenzyl)sulphide, benzoic esters (parabens) likemethylparaben, propylparaben, butylparaben, ethylparaben,isopropylparaben, isobutylparaben, benzylparaben, sodium methylparaben,and sodium propylparaben, halogenated carbanilides (e.g.,3,4,4′-trichlorocarbanilides (TRICLOCARBAN® or TCC),3-trifluoromethyl-4,4′-dichlorocarbanilide, 3,3′,4-trichlorocarbanilide,etc.), cationic actives such as benzalkonium chloride, and clotrimazole.Another class of antimicrobial agents (specifically antibacterialagents) which are useful, are the so-called “natural” antibacterialactives, referred to as natural essential oils. Typical naturalessential oil antibacterial actives include oils of anise, lemon,orange, rosemary, wintergreen, thyme, lavender, cloves, hops, tea tree,citronella, wheat, barley, lemongrass, cedar leaf, cedarwood, cinnamon,fleagrass, geranium, sandalwood, violet, cranberry, eucalyptus, vervain,peppermint, gum benzoin, basil, fennel, fir, balsam, menthol, ocmeaoriganum, Hydastis carradensis, Berberidaceae daceae, Ratanhiae andCurcuma longa.

The cleaning formulations may be packaged in a variety of suitablepackaging known to those skilled in the art. The liquid formulations maydesirably be packaged in manually operated spray dispensing containers,which are usually made of synthetic organic polymeric plastic materials.Accordingly, disinfecting formulations containing the compounds andpackaged in a spray dispenser, preferably in a trigger spray dispenseror a pump spray dispenser, are envisioned. Spray-type dispensers allowto uniformly apply to a relatively large area of a surface to bedisinfected a liquid cleaning formulations described herein.

The compounds can be impregnated into a nonwoven absorbent wipe.Disinfectant wet wipes are also disclosed for example in U.S. Pat. No.8,563,017.

The compounds can be in an aqueous foam with a special surfactant systemcapable of generating a foam. See U.S. Pat. No. 8,097,265, U.S. Pat. No.5,891,922 and U.S. Pat. No. 4,889,645.

The compounds can also be in a pressurized spray aerosol. See also, U.S.Publication No. 20010053333 which discloses a liquid flash-dry aerosoldisinfectant composition with a flash vaporization component and aneffective amount of an antimicrobial agent.

It is within the abilities of one of ordinary skill in the art todetermine the effective amount of the compounds to include in anaerosol, foam, solution or disinfectant cloth for the purpose ofsterilizing for example, high risk hospital surfaces.

D. Conjugation and Immobilization of Compounds

The one or more compounds as defined in section III.A above for use inaccordance with any of the first, second or third aspect of the presentinvention and may be presented as conjugated and/or immobilizedcompounds.

The compounds may be conjugated with other agents in order to retain thecompounds on surfaces, for example, to prevent biofilm formation on asurface. In one embodiment, the compounds may be conjugated to an agentthat has affinity for a surface in order to retain the compounds on thatsurface. For example, the compounds may be conjugated to an agentwherein the agent is a polymer or oligomer, and the polymer or oligomerhas a high affinity for the surface.

In another embodiment the compounds may be conjugated to an agentwherein the agent comprises a reactive moiety suitable for anchoring toa surface. The reactive moiety may, for example, be photo-reactive, orcapable of coupling covalently to a surface. The reactive moiety mayalso incorporate spacers and linkers and other functional groups inorder to place the compound in a desired location relative to thesurface. FIGS. 15A-C are examples of how FeQ (Fe-QA) may be conjugatedto an agent comprising a reactive moiety suitable for anchoring to asurface. In each of the three examples, FeQ is conjugated to a calix [4]arene frame that comprises a reactive moiety. In FIG. 15A, FeQ isconjugated via a linker to a calix [4] arene frame that contains aphotoreactive functional group. FIG. 15B is a variant of FIG. 15A whichshows that the reactive moiety can be positioned at a different locationon the calix [4] arene frame. FIG. 15C is an example of FeQ conjugatedto a calix [4] arene frame, wherein the latter is functionalized withthiol groups that are capable of reacting with surfaces. It should beunderstood that different linkers or no linkers may be used, and thatother agents may be used instead of the calix [4] arene frame, includingcyclodextrins and other polymers and oligomers.

In yet another embodiment, the compounds may be conjugated to an agentthat comprises a substance with an affinity for a surface. The agent mayincorporate spacers and linkers and other functional groups in order toplace the compound in the desired location relative to the surface. Inone embodiment, the agent contains hydroxyapatite. FIGS. 16 A and B areexamples of how FeQ (Fe-QA) may be conjugated via a linker tohydroxyapatite. In these examples, the linkers are attached in differentpositions to one of the quinic acid ligands via a functional group, Y′,and at the other end of the linker are attached to hydroxyapatite (HA)via a second functional group, X′. In an alternative embodiment, the HAgroup in the structures of FIGS. 16A and B may be replaced with areactive group that can attach (or be attached) to a surface, such as aphoto-reactive compound, isocyanate, hydroxy group, amine,trialkoxysilyl ether, such as a triethoxysilyl ether, or phosphateester. These groups may be attached directly to the polyethylene glycol,or an additional linker inserted between the reactive group and thepolyethylene glycol.

E. Feeds and Feed Supplements

In accordance with the first aspect of the present invention, a furtherembodiment of the present invention provides that the compounds asdefined in section III.A can be formulated into growth promotingformulations.

The one or more compounds may be used, for example, in feed or formulato improve the growth of chicken, for example, a meat-type chicken suchas broiler chicken, or an egg-laying chicken such as a pullet or hen, ora breeder chicken, other poultry, such as a turkey, geese, quail,pheasant, or ducks, or livestock such as cattle, sheep, goats, swine,alpaca, banteng, bison, camel, cat, deer, dog, donkey, gayal, guineapig, horse, llama, mule, rabbit, reindeer, water buffalo, yak, althoughthe skilled person will appreciate that other feeds for animals,including zoo animals, captive animals, game animals, fish (includefreshwater and saltwater fish, farmed fish, and ornamental fish), othermarine and aquatic animals, including shellfish such as, but not limitedto, oysters, mussels, clams, shrimps, prawns, lobsters, crayfish, crabs,cuttlefish, octopus, and squid, domestic animals such as cats and dogs,rodents (such as mice, rats, guinea pigs, hamsters), and horses, arealso provided, as well as any other domestic, wild and farmed animal,including mammals, marine animals, amphibians, birds, reptiles, insectsand other invertebrates. The one or more compounds may be added todrinking water for any of said animals to improve growth

The compounds may be useful in treatment of ponds, tanks, or otheraquatic or marine environments containing fish (include freshwater andsaltwater fish, farmed fish and ornamental fish), other marine andaquatic animals, including shellfish or crustaceans such as shrimp,oysters, mussels, clams, prawns, lobsters, crayfish, crabs, cuttlefish,octopus and crawfish.

The one or more compounds may be used alone or in combination with otheranti-microbial, bactericidal or bacteriostatic compounds (for example,in accordance with the second or third aspect of the present invention)and/or growth enhancing agents.

The compounds as defined in section III.A can improve growthperformance, and can be used to increase average body weight duringgrowth. The compounds can also be used to improve feed conversion ratio.In particular, the compounds can be used to decrease the mortalityadjusted feed conversion ratios (MFCR). The compounds may be used toproduce animals with higher average body weight in a given period oftime, or may be used to reach a target average body weight in a shorterperiod of time. The compounds may be used to decrease the amount of feednecessary for an animal to attain a target weight. In addition, thecompounds may be used in stressed environments to improve growth andMFCR. These environments include but are not limited to high stockingdensities of animals, dirty pen litter, presence of pathogens, presenceof Campylobacter and other bacteria, and high temperature environments.

The compositions are particularly useful in feeds for commercial birdssuch as chickens, turkeys, pheasants, and ducks. Exemplary poultry feedsin which the as one or more compounds defined in section III.A can beincluded, include poultry feeds that are referred to as “complete”feeds, because they are designed to contain all the protein, energy,vitamins, minerals, and other nutrients necessary for proper growth, eggproduction, and health of the birds. Feeding any other ingredients,mixed with the feed or fed separately, upsets the balance of nutrientsin the “complete” feed. Feeding additional grain or supplement with thecomplete poultry feed is not recommended.

Chickens used in optimized commercial broiler production are typicallyfed different diets depending upon their age. For example, chickens forbroiler production may be raised using three diets. These diets aretypically called a “starter”, “grower” and “finisher”. The starter dietmay be fed for about the first 10-12 days (typically in the range of7-14 days). This starter diet is followed by the grower diet, which isprovided to the broilers for almost 2 weeks (typically from about 11-24days). The finisher diet is used for the remainder of the productionperiod (typically from 24 to 42 days). Some broiler houses will use moreor less diets (for example 4 diets), and vary the timing of dietchanges. Broilers are typically harvested between 30 and 42 days,although this time can be longer or shorter. Further details and optionsare discussed above in the context of the first aspect of the presentinvention.

F. Treatment to Promote Growth

As discussed above in more detail, in the context of the first aspect ofthe present invention, it has been discovered that the one or morecompounds defined in section III.A of this application, above, areparticularly useful in promoting growth. The compounds may be added toanimal feed or animal drinking water in order to promote growth.Addition of the compounds to feed or drinking water results in improvedgrowth. It has also been discovered that the compounds can be added toanimal feed or animal drinking water in order to decrease the mortalityadjusted feed conversion ratio. Thus it is possible to use the compoundsto decrease the amount of feed necessary for an animal to grow. Thecompounds may further be administered with other animal additives, andmay be administered in commercial feeds. In a preferred embodiment, thecompounds are administered in feeds.

It has also been discovered that the compounds can be administered toanimals that are in a stressed environment in order to improve theirgrowth performance. In a stressed environment the compounds promotegrowth that yields animals with higher average body weights. Thecompounds also decrease mortality adjusted feed conversion ratios instressed environments.

EXAMPLES

The following non-limiting examples are included to demonstrateparticular embodiments of the various aspects of the present invention.It should be appreciated by those of skill in the art that thetechniques disclosed in the examples which follow represent techniquesdiscovered by the inventor to function well in the practice of theinvention, and thus can be considered to constitute preferred modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1. Inhibition of Biofilm Formation on Beads Surface byEnterococcus faecalis NCTC 12697 Using Fe-QA

Materials and Methods

Bacteria (Enterococcus faecalis NCTC 12697, Staphylococcus epidermidisF1513 and Staphylococcus aureus ATCC 25923) were grown on Brain heartinfusion (BHI) passaged to new medium either containing Fe-QA or alone.Growth suspensions were prepared at 0.0001 OD/ml and then allowed togrow at 37° C. under normal atmospheric conditions for 24 h in BHI withplastic coated UV beads (Lascells). After 48 h, 10 μl suspension wasserially diluted 10 fold to 10-3, 10-4, 10-5, 10-6, 10-7, 10-8. For eachdilution, 10 μd was spotted on BHI agar plates and colonies countedafter 24 h. The beads were also removed washed in PBS before adding to 1ml PBS. After vortex mixing, 10 μl of the cell suspension was seriallydiluted as above and cell counts carried out.

Results

Enterococcus faecalis causes many of the antibiotic resistant infectionsin hospitals, a consequence of its inherent resistance to certainantibiotics and

of its ability to survive and proliferate in the intestinal tract. ASer/Thr kinase in Enterococcus faecalis is found to mediateantimicrobial resistance. Studies have shown that PrkC, a one-componentsignaling protein containing a eukaryotic-type Ser/Thr kinase domain,allowed for inherent antimicrobial resistance and intestinal persistenceof E. faecalis (Kristich, et al., Proc. Nat. Acad. Sci. USA,104(9):3508-3513 (2007)). Kristich, et al. found that an E. faecalismutant lacking PrkC grew at a wild-type rate in the absence ofantimicrobial stress but showed enhanced sensitivity tocell-envelope-active compounds, including antibiotics that targetedcell-wall biogenesis and bile detergents. PrkC regulates physiologicalprocesses in E. faecalis that are key to its success as a nosocomialpathogen.

The effect of Fe-QA on biofilm formation by E. faecalis was tested asdescribed in the materials and methods. The data (FIGS. 1A and 1B) showsthat Fe-QA inhibited E. faecalis biofilm formation as measured followingtreatment of E. faecalis grown on plastic coated UV beads.

Example 2. Inhibition of Biofilm Formation on Beads Surface byStaphylococcus epidermidis F1513 Using Fe-QA

Materials and Methods

The effect of Fe-QA on biofilm formation by S. epidermidis F1513 wastested as described in the materials and methods of Example 1.

Results

The data (FIGS. 2A and 2B) shows that Fe-QA inhibited S. epidermidisbiofilm formation as measured following treatment of S. epidermidisgrown on plastic coated UV beads.

Example 3. Inhibition of Biofilm Formation on Beads Surface byStaphylococcus aureus ATCC 25923 Using Fe-QA

The treatment of choice for S. aureus infection is penicillin; in mostcountries, however, penicillin resistance is extremely common, andfirst-line therapy is most commonly a penicillinase-resistant β-lactamantibiotic (for example, oxacillin or flucloxacillin). Combinationtherapy with gentamicin may be used to treat serious infections, such asendocarditis, but its use is controversial because of the high risk ofdamage to the kidneys (Cosgrove, et al., Clin Infect Dis, 48(6):713-721(2009). The duration of treatment depends on the site of infection andon severity.

Materials and Methods

The effect of Fe-QA on biofilm formation by S. aureus was tested asdescribed in the materials and methods of Example 1.

Results

The data (FIGS. 3A and 3B) shows that Fe-QA inhibited S. aureus biofilmformation as measured following treatment of S. aureus grown on plasticcoated UV beads.

Example 4. Phenotypic Changes and Inhibition of Campylobacter jejuniNCTC 11168 Binding to Histo-Blood Group Antigens by Fe-QA

Materials and Methods

Binding of C. jejuni NCTC 11168 to the BgAgs (common ABO histo-bloodgroup antigens), Le^(b) and H-II was measured after growing the bacteriain a medium that has either Fe-QA at 0.34 mM or 3.4 mM. The binding ismeasured by washing away the Fe-Q containing medium prior to testing thebacteria by ELISA as described below. Binding was measured after onepassage and four passages (4 generations) with Fe-QA included in themedium, and compared to a control without Fe-QA.

Binding of C. jejuni 11168 to BgAgs (common ABO histo-blood groupantigens) that are expressed, for example, on the surfaces oferythrocytes, and the inhibition of this binding by Fe-QA was quantifiedusing the ELISA method described below.

The ELISA was performed as follows: BSA-BgAg conjugates were obtainedfrom IsoSep, Tullinge, Sweden. Coupling of BgAgs to 96-well plates (NUNCImmobilizer Amino) was carried out by the addition of 100 μl BSA-BgAg (5μg/ml unless stated otherwise) in sodium carbonate buffer to each well.Plates were incubated at room temperature for 2 h before unbound reagentwas removed by washing three times in PBS-T. All wells were blocked bythe addition of 100 ml 1% BSA/PBS and incubated for 2 h at roomtemperature. After further washing in PBS-T, 100 ml of DIG-taggedbacteria (at OD₆₀₀ of 0.05) were added to each well and incubatedovernight at 4° C. Plates were washed three times in PBS-T before 100 mlanti-digoxigenin-POD solution (Roche Diagnostics; 1 in 5000 diluted in1% BSA/PBS) was added and incubated for 1 h at room temperature. Plateswere again vigorously washed in PBS-T and color developed by adding 100μl ABTS substrate (Roche). Plates were read with an ELISA reader (BiotekEL800) at an absorbance of 405 nm. Specific binding was determined bysubtracting the binding of each strain to BSA (typically OD4050.07-0.09) from the binding to each BSA-BgAg conjugate. Inhibitionassays shown in FIG. 4B were carried out as above but after the removalof the blocking solution, DIG-labeled C. jejuni was pre-incubated for 4h with an Fe-QA solution (0.34 mM) before being added to each well.

Results

FIG. 4A shows the binding of C. jejuni to the BgAgs (common ABOhisto-blood group antigens), Le^(b) and H-II, after growing the bacteriain a medium that has either Fe-QA at 0.34 mM or 3.4 mM. The results showa marked decrease in binding to Le^(b) and H-II particularly in thegroup that was treated with the higher concentration of Fe-QA (3.4 mM).A statistically significant decrease was also found at the lower Fe-QAconcentration of 0.34 mM when compared to the control group. It istherefore apparent that treating the bacteria with Fe-QA for severalgenerations results in a phenotypic change, and the bacteria lose theability to bind to the BgAg's permanently.

FIG. 4B shows the binding of C. jejuni 11168 to BgAgs (common ABOhisto-blood group antigens) that are expressed, for example, on thesurfaces of erythrocytes, and the inhibition of this binding by Fe-QA.Binding is quantified using the ELISA method described below. The bargraphs show binding for the non-treated C. jejuni 11168-NT(NT=non-treated) to BgAgs, and the significant inhibition of binding bypre-incubation of the bacteria with Fe-QA prior to adding the bacteriato the ELISA plate.

Example 5. Inhibition of Helicobacter pylori CCUG 17875 Attachment toHuman Gastric Tissue

Materials and Methods

The ability of H. pylori CCUG 17875 to bind to samples of human gastrictissue in the presence of Lewis b antigen, Le^(b), and twoconcentrations of Fe-QA (1 mM and 0.2 mM) was measured. Binding wasquantified by fluorescence using H. pylori that had been labeled withfluorescein using FITC (fluorescein isothiocyanate), and human gastrictissue that was embedded in parafilm. The bacteria were suspended inblocking buffer (1% BSA in PBS) and applied to re-hydratedhisto-sections of human gastric tissue. Binding was assessedmicroscopically, and quantified as the average number of bacteria boundto the tissue.

Binding of Le^(b) to H. pylori strain 17875 at pH 7.4 was assayed bylabeling Le^(b) with radioactive iodine (1-125), mixing the radioactiveantigen with H. pylori bacteria, pelletizing the bacteria using acentrifuge, and measuring the radioactivity in the pellet and in thesupernatant. Any Le^(b) that is bound to the bacteria is quantified bythe measurement of radioactivity in the pellet. The ratio of theradioactivity in the pellet to the supernatant therefore corresponds tothe ratio of Le^(b) bound by the bacteria to that which remains unbound,expressed as bound/free.

Results

The ability of H. pylori to bind to samples of human gastric tissue inthe presence of Lewis b antigen, Le^(b), and two concentrations of Fe-QA(1 mM and 0.2 mM) is shown in FIG. 5A The results show that the bindingof H. pylori to human gastric tissue is significantly reduced in thepresence of Le^(b) (10 μg/ml) and Fe-QA at both 1 mM and 0.2 mMconcentrations when incubated for 1 hour at room temperature. Reductionin bacterial binding was estimated by counting the number ofspecifically adhered bacteria to the gastric pit region under 200×magnification. Fe-QA therefore prevents bacterial attachment of H.pylori to gastric epithelium.

FIG. 5B shows the competitive inhibition of Le^(b) binding to H. pyloriby Fe-QA as the concentration of Fe-QA is increased. The graph is a plotof the ratio of bound/free Le^(b) versus Fe-QA concentration (μM). Thegraph shows that Fe-QA increasingly inhibits binding of the Le^(b) to H.pylori as the amount of Fe-QA is increased.

Example 6. Fe-QA Prevention of Biofilm Formation by Pseudomonasaeruginosa and Uropathogenic E. coli (UPEC)

Materials and Methods

Pseudomonas aeruginosa PAO-1, and a clinically isolated uropathogenicEscherichia coli UPEC-536 were routinely grown on either LB(Luria-Bertani, Oxoid, UK) agar plates at 37° C. or in broth at 37° C.with 200 rpm shaking. UV-sterilized glass slides were incubated ineither 15 mL RPMI-1640 defined medium (Sigma, UK) or 15 mL RPMI-1640with Fe-QA inoculated with diluted (OD₆₀₀=0.01) bacteria from overnightcultures at 37° C. with 60 rpm shaking for 72 hours. The slides wereremoved from bacterial culture and washed with 15 mL phosphate bufferedsaline at room temperature for 5 minutes three times and then rinsedwith distilled H₂O. After washing, the slides were stained with 20 μMSYTO17 dye (Invitrogen, UK) at room temperature for 30 minutes. Afterremoving excess staining dye and air-drying, the samples were examinedusing a Carl Zeiss LSM 700 Laser Scanning Microscope with ZEN 2009imaging software (Carl Zeiss, Germany). The coverage rate of bacteria onthe surface was analysed using open source Image J 1.44 software(National Institute of Health, US).

Results

FIG. 6A shows that Fe-QA (“X”) at 100 μM inhibits the formation ofbiofilm by Pseudomonas aeruginosa. In the absence of Fe-QA, a highercoverage rate was measured for Pseudomonas aeruginosa than in thepresence of a 100 μM concentration of Fe-QA.

FIG. 6B shows that Fe-QA inhibits the formation of biofilm byUropathogenic E. coli (UPEC). In the absence of Fe-Q (“0 μM), a highercoverage rate is measured for UPEC than in the presence of 0.1 μM, 1 μM,10 μM and 100 μM concentrations of Fe-QA.

Example 7. Planktonic Growth of Bacteria in the Presence of Fe-QA

Materials and Methods

The growth rate of Uropathogenic E. coli UPEC-536 in RPMI-1640 mediaover a period of 24 hours was compared to the growth rate of UPEC inRPMI-1640 media, but in the presence of 100 μM Fe-QA. The growth rate ofPseudomonas aeruginosa in RPMI-1640 media was also compared to thegrowth rate of Pseudomonas aeruginosa in RPMI-1640 media, but in thepresence of 100 μM Fe-QA.

Results

FIG. 7A is a graph showing the growth rate of UPEC in RPMI-1640 mediaover a period of 24 hours. The growth rate is compared to the growthrate of UPEC in RPMI-1640 media, but in the presence of 100 μM Fe-QA.(The optical absorbance of the RPMI-1640 is also shown for reference.)The graph demonstrates that Fe-QA does not inhibit the growth of UPEC.However, as shown in Example 6 and FIG. 6B, Fe-QA inhibits biofilmformation. Therefore, the inhibition of biofilm formation is not due tobacterial growth inhibition.

FIG. 7B is a graph showing the growth rate of Pseudomonas aeruginosa inRPMI-1640 media. The growth rate is compared to the growth rate ofPseudomonas aeruginosa in RPMI-1640 media, but in the presence of 100 μMFe-QA. (The optical absorbance of the RPMI-1640 is also shown forreference.) The graph demonstrates that Fe-QA does not inhibit thegrowth of Pseudomonas aeruginosa as was also found for UPEC (above).However, as shown in Example 6 and FIG. 6A, Fe-QA inhibits biofilmformation of Pseudomonas aeruginosa. Therefore, the inhibition ofbiofilm formation is not due to bacterial growth inhibition.

Example 8. Planktonic Growth Versus Biofilm Growth of C. jejuni MOMPT268G Mutant

Materials and Methods

A MOMP-T strain of Campylobacter jejuni NCTC 11168 was prepared bymutating T268 of MOMP. The T268 of MOMP was replaced with glycine. Theplanktonic growth of the MOMP-T strain of Campylobacter jejuni and itsability to form a biofilm compared to the wild type strain wasdetermined. Bacteria (wildtype and the MOMP-T strain) were grown on CCDAselective charcoal agar and then passaged to Mueller-Hinton broth (MHB).Growth suspensions were prepared at 0.02 OD/ml and then allowed to growat 37° C. under atmospheric conditions (5% CO₂, 6% O₂) for 48 h in MHBwith plastic coated UV beads (Lascells). After 48 h, 10 μl suspensionwas serially diluted 10 fold to 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸.For each dilution, 10 μl was spotted on CCDA selective agar plates andcolonies counted after 48 h. The beads were also removed and washed inPBS before adding to 1 ml PBS. After vortex mixing, 10 μl of the cellsuspension was serially diluted as above and cell counts carried out toquantify biofilm formation.

Results

FIG. 8A shows that mutation of T268 of MOMP does not impact the growthof Campylobacter jejuni relative to wildtype even though it is knownthat T268 of MOMP is O-glycosylated. However, FIG. 8B shows thatmutation of T268 of MOMP does impact biofilm formation. The experimentdemonstrates that O-glycosylation of MOMP at T268 in Campylobacter isvital for biofilm formation, and the necessity of the MOMP-T268 glycanfor aggregation and biofilm formation.

Example 9. Fe-QA Treatment Makes Antibiotic Resistant Strain of E. coliLose Resistance to Antibiotics

Materials and Methods

A kanamycin resistance gene was introduced into the genome ofEnteropathogenic E. coli EPEC, (E2348/69), genotype; WildtypeEPEC-O-17:H6 allowing the kanamycin resistant strain of EPEC to grow inthe presence of a patch that has been immersed in kanamycin (at aconcentration of 50 μg/mL). The kanamycin resistant strain of EPEC wasthen immersed in a patch in a solution of Fe-QA.

Results

Immersing a patch in a solution of Fe-QA, and then placing the patch ona growth plate with the kanamycin resistant strain also had nosignificant effect on bacterial growth. However, immersing the patch ina solution of Fe-QA and kanamycin, and then placing on a growth platecaused the bacteria to fail to grow. The experiment demonstrates thatFe-QA has no significant impact on the growth of the bacteria. However,Fe-QA makes the bacteria more sensitive to kanamycin even though thebacteria carry a kanamycin resistant gene, and is therefore resistant tothe antibiotic. The Fe-QA can therefore be used in conjunction withantibiotics to kill or inhibit the growth of antibiotic resistantbacteria.

FIG. 9 is a graph that shows the impact on the growth curve of the EPECkanamycin resistant strain when grown in the presence of: (i) kanamycin(line number 2 from top, (triangles)), (ii) Fe-QA (line number 1, fromtop), and (iii) kanamycin and Fe-QA (line number 3, from top (squares)).The most rapid growth was found when the strain was grown in medium onlycontaining Fe-QA. However, a difference was observed between the rate ofgrowth of the strain in the presence of kanamycin versus kanamycin andFe-QA. The double-headed arrow indicates the difference in rates betweenthese two growth conditions. The rate of growth of the strain wasinhibited in the presence of kanamycin and Fe-Q relative to the rate ofgrowth of the strain just in the presence of kanamycin. This is furtherevidence that Fe-QA can be used in conjunction with antibiotics to killor inhibit the growth of antibiotic resistant bacteria.

Example 10. Fe-QA Treatment Makes Antibiotic Resistant Strain of C.jejuni NCYC 11168 Lose Resistance to Antibiotic

Materials and Methods

A patch immersed in a solution of Fe-QA was placed on a growth platewith wildtype Campylobacter jejuni NCTC 11168 (that does not carry akanamycin resistance gene). The wildtype C. jejuni grew normally, andwas not affected by FeQ. A patch was also immersed in a solution ofkanamycin and Fe-QA, and placed on a growth plate of wildtype C. jejuni.The growth of C. jejuni was impacted showing that wildtype C. jejuni issensitive to the presence of kanamycin.

The patch experiments were repeated with a Campylobacter jejuni NCTC11168 strain carrying a kanamycin resistance gene wherein patches wereimmersed in (i) kanamycin only, (ii) Fe-QA only, and (iii) kanamycin andFe-QA. The growth of the C. jejuni strain carrying the kanamycinresistance gene was not impacted by the patches immersed in (i)kanamycin and (ii) Fe-QA, however, the growth was impacted by the patchthat was immersed in both kanamycin and Fe-QA. The results furtherdemonstrate that Fe-QA can be used in combination with antibiotics toinhibit or kill antibiotic resistant bacteria.

Example 11. Fe-Tyr Treatment Makes Antibiotic Resistant Strain ofEnteropathogenic E. coli (EPEC) E2348/69 Lose Resistance to Antibiotic

Materials and Methods

The impact on the growth curve of antibiotic resistant EnteropathogenicE. coli (EPEC) strain E2348/69 (genotype Wild Type EPEC O17:H6) whengrown in the presence of (i) gentamicin (1.25 μM), (ii) Fe-Tyr (100 μM),(iii) gentamicin (1.25 μM) and Fe-Tyr (1.25 μM), and (iv) a control withno gentamicin or Fe-Tyr present was determined.

Results

FIG. 10A is a graph that shows the impact on the growth curve ofantibiotic resistant Enteropathogenic E. coli (EPEC) strain E2348/69(genotype Wild Type EPEC O17:H6) when grown in the presence of (i)gentamicin (1.25 μM) (grey circles), (ii) Fe-Tyr (100 μM) (grey invertedtriangles), (iii) gentamicin (1.25 μM) and Fe-Tyr (1.25 μM) (whiteupright triangles), and (iv) a control with no gentamicin or Fe-Tyrpresent (black circles). The most rapid growth (see FIG. 10B) was foundwhen the strain was grown in medium only containing Fe-Tyr, which wascomparable to the control (without gentamicin or Fe-Tyr present).However, a difference was observed between the rate of growth of thestrain in the presence of the antibiotic gentamicin versus gentamicinand Fe-Tyr. Thus it was shown that the rate of growth of the strain wasinhibited in the presence of gentamicin and Fe-Tyr relative to the rateof growth of the strain just in the presence of gentamicin. This isevidence that Fe-Tyr can be used in conjunction with antibiotics to killor inhibit the growth of antibiotic resistant bacteria.

Example 12. FeQ (Known Also as “Fe-QA”) Treatment Makes AntibioticResistant Strain of Enteropathogenic E. coli (EPEC) E2348/69 LoseResistance to Antibiotic

Materials and Methods

The impact on the growth curve of antibiotic resistant EnteropathogenicE. coli (EPEC) strain E2348/69 (genotype Wild Type EPEC O17:H6) whengrown in the presence of a fixed concentration of gentamicin (1.25 μM)and an increasing concentration of FeQ versus the strain grown in thepresence of only FeQ or only gentamicin, was determined.

Results

FIGS. 11A-C are graphs that show the impact on the growth curve ofantibiotic resistant Enteropathogenic E. coli (EPEC) strain E2348/69(genotype Wild Type EPEC O17:H6) when grown in the presence of a fixedconcentration of gentamicin (1.25 μM) and an increasing concentration ofFeQ (FIG. 11A: 10 μM, FIG. 11B: 34 μM and FIG. 11C: 68 μM) versus thestrain grown in the presence of only FeQ or only gentamicin. As shown inFIG. 11D, increasing the concentration of FeQ from 10 μM to 100 μM didnot impact the growth rate of the strain. However, FIG. 11E clearlyshows that a difference was observed between the rate of growth of thestrain in the presence of the antibiotic gentamicin versus thecombination of gentamicin and FeQ. Thus it was shown the rate of growthof the strain was inhibited in the presence of gentamicin and FeQrelative to the rate of growth of the strain just in the presence ofgentamicin. This is further evidence that FeQ can be used in conjunctionwith antibiotics to kill or inhibit the growth of antibiotic resistantbacteria.

Example 13. FeQ Prevents Attachment of Bacteria to Surfaces

Materials and Methods

Enteropathogenic E. coli (EPEC) E2348/69 were grown in wells for 48hours at 37° C. in the presence of FeQ (100 μM), and in the absence ofFeQ (as control). After 48 hours, the wells were washed in order toremove suspended cells. Crystal violet was then added to each well. Thewells were then washed to remove excess dye. A mixture ofacetone/ethanol was then added to the wells to re-suspend any cellsattached to the plastic surface of the wells, and dissolve any dyepresent. The presence of dye in each well was then quantified bymeasuring the O.D. at 570 nm.

Results

In the absence of FeQ, EPEC binds to the plastic surface and forms abiofilm that is readily detected by dying with crystal violet. However,in the presence of FeQ, EPEC is unable to attach to the plastic surfaceand form a biofilm, and is not detected by adding crystal violet. FIG.12 shows quantitatively the difference in the attachment of EPEC cellsto the plastic well surface in the absence and presence of FeQ bymeasurement of the optical absorbance of crystal violet that wasabsorbed by EPEC cells attached to the surface. At an FeQ concentrationof 100 μM there is little or no attachment of bacterial cells to thesurface and no biofilm formation.

Example 14. FeQ (Known Also as “Fe-QA”) Treatment Makes AntibioticResistant Strain of Pseudomonas aeruginosa PAO-1 Lose Resistance toAntibiotic

Materials and Methods

The impact on the growth curve of an antibiotic resistant clinicalisolate of Pseudomonas aeruginosa (PAO-1 Clinical) grown in the presenceof kanamycin and FeQ versus the clinical isolate grown in the presenceof (i) FeQ, (ii) kanamycin or (iii) without addition of FeQ or kanamycin(control) was assessed.

Results

FIG. 13 is a graph that shows the impact on the growth curve of anantibiotic resistant clinical isolate of Pseudomonas (PAO-1 Clinical)grown in the presence of kanamycin and FeQ versus the clinical isolategrown in the presence of (i) FeQ, (ii) kanamycin or (iii) withoutaddition of FeQ or kanamycin (control). The graph shows a largereduction in the growth rate of the Pseudomonas PAO clinical isolatewhen kanamycin and FeQ are added to the growth media versus eitherkanamycin or FeQ alone. The example demonstrates that FeQ causes thePseudomonas clinical isolate to lose its sensitivity to the antibiotic.

Example 15. Impact of FeQ and Mutation of the Glycosylation Site of theMOMP Protein of Campylobacter jejuni on Protein Expression

Materials and Methods

In order to determine the role of FeQ and glycosylation on proteinexpression by Campylobacter, a quantitative proteomic approach wasundertaken to determine protein contents upon FeQ treatment ofCampylobacter jejuni NCTC 11168 wildtype (WT) and a mutant strain(MOMP^(T268G)), also referred to as MOMP-T, where the O-glycosylationsite of MOMP had been disrupted by an amino acid substitution preventingglycosylation of MOMP. Protein expression of (i) Campylobacter jejuni11168 wildtype (WT), (ii) Campylobacter jejuni 11168 MOMP-T (MOMP-T),(iii) Campylobacter jejuni 11168 wildtype (WT) in the presence of FeQ,and (iv) Campylobacter jejuni 11168 MOMP-T in the presence of FeQ weredetermined using isobaric tags for relative and absolute quantitation(iTRAQ). Bacterial proteins were identified using LC-MS/MS and iTRAQ,and functions assigned according to the Gene Ontology classificationsystem. A total of 274,533 mass spectra from all samples led to theidentification of 626 peptides (i.e. 420 unique peptides that wereassigned to 160 Campylobacter proteins).

Results

Table 1 shows the results of iTRAQ analysis comparing the ratios ofprotein expression for (a) WT/MOMP-T, (b) WT+FeQ/WT, (c)MOMP-T+FeQ/MOMP-T, and (d) WT+FeQ/MOMP-T+FeQ, wherein (a) is the ratioof Campylobacter jejuni 11168 wildtype to the Campylobacter jejuni 11168T268G mutant, (b) is the ratio of Campylobacter jejuni 11168 wildtypetreated with FeQ to Campylobacter jejuni 11168 wildtype, (c) is theratio of the Campylobacter jejuni 11168 T268G mutant treated with FeQ tothe Campylobacter jejuni 11168 T268G mutant, and (d) is the ratio ofCampylobacter jejuni 11168 wildtype treated with FeQ to theCampylobacter jejuni 11168 T268G mutant treated with FeQ.

TABLE 1 iTRAQ analysis MOMP- WT + FeQ/MOMP- WT/MOMP-T WT + FeQ/WT T +FeQ/MOMP-T T + FeQ Accession Description MW [kDa] Coverage # Proteins #Unique Peptides # Peptides 114/116 115/114 117/116 115/117 A0A059H879Molecular chaperone GroEL 57.9 56.51 30 20 24 0.821 1.460 1.222 1.082 OS= Campylobacter jejuni Cj1 GN = groEL PE = 4 SV = 1 - [A0A059H879_CAMJU]A3ZEC2 Elongation factor Tu 43.6 51.63 16 14 15 0.916 0.907 0.945 0.949OS = Campylobacter jejuni subsp. jejuni HB93-13 GN = tuf PE = 3 SV = 1 -[A3ZEC2_CAMJU] H7YTU2 Putative GMC 63.7 27.23 34 11 12 1.494 1.476 1.3191.894 oxidoreductase subunit OS = Campylobacter jejuni subsp. jejuni LMG23357 GN = cje133_09008 PE = 4 SV = 1 - [H7YTU2_CAMJU] H7YEB5Nickel-dependent 63.5 30.30 36 9 12 0.585 1.027 0.890 0.818 hydrogenase,large subunit OS = Campylobacter jejuni subsp. jejuni LMG 9879 GN =cje120_00450 PE = 3 SV = 1 - [H7YEB5_CAMJU] A0A059GL54 Cytochrome Cbiogenesis 36.8 34.90 18 9 11 1.162 0.694 0.778 1.118 protein CcsA OS =Campylobacter jejuni 255 GN = L034_08235 PE = 4 SV = 1 -[A0A059GL54_CAMJU] H7ZE54 Chemotaxis protein CheA 85.2 20.03 24 9 130.720 1.176 1.034 0.861 OS = Campylobacter jejuni subsp. jejuni2008-1025 GN = cje145_01162 PE = 4 SV = 1 - [H7ZE54_CAMJU] E5Z9V4Methyl-accepting chemotaxis 71.3 38.73 229 8 21 0.893 1.172 1.049 1.105protein (MCP) signaling domain protein (Fragment) OS = Campylobacterjejuni subsp. jejuni DFVF1099 GN = CSQ_1786 PE = 4 SV = 1 -[E5Z9V4_CAMJU] A0A023WIW7 Fumarate reductase 73.7 19.76 30 8 11 0.8650.832 0.766 0.992 flavoprotein subunit OS = Campylobacter jejuni subsp.jejuni CG8421 GN = CJ8421_02050 PE = 4 SV = 1 - [A0A023WIW7_CAMJU]W8JBW7 Cytochrome C 41.4 29.95 25 8 10 0.574 1.116 0.820 0.816 OS =Campylobacter jejuni subsp. jejuni NCTC 11168-Kf1 GN = N919_05910 PE = 4SV = 1 - [W8JBW7_CAMJE] A8FJR2 ATP synthase subunit beta 50.8 20.65 14 78 0.718 1.106 0.942 1.019 OS = Campylobacter jejuni subsp. jejuniserotype O:6 (strain 81116/NCTC 11828) GN = atpD PE = 3 SV = 1 -[ATPB_CAMJ8] Q9PPE0 Probable thiol peroxidase 18.4 60.00 12 7 8 1.1591.281 1.162 1.446 OS = Campylobacter jejuni subsp. jejuni serotype O:2(strain NCTC 11168) GN = tpx PE = 3 SV = 1 - [TPX_CAMJE] E5ZFX0Methyl-accepting chemotaxis 41.8 26.39 95 7 8 0.676 1.183 1.061 0.748protein (MCP) signaling domain protein (Fragment) OS = Campylobacterjejuni subsp. jejuni 305 GN = CSS_2003 PE = 4 SV = 1 - [E5ZFX0_CAMJU]H7ZH37 Putative amino-acid 30.9 46.95 29 7 12 0.568 1.623 1.058 0.980transporter periplasmic solute-binding protein OS = Campylobacter jejunisubsp. jejuni 2008-1025 GN = cje145_06564 PE = 4 SV = 1 - [H7ZH37_CAMJU]D2MUR2 Possible bacterioferritin 17.2 69.80 14 7 9 0.766 1.045 0.9510.939 OS = Campylobacter jejuni subsp. jejuni 1336 GN = C1336_000320070PE = 3 SV = 1 - [D2MUR2_CAMJU] H8BN80 Methyl-accepting chemotaxis 76.121.16 79 6 10 0.702 1.106 1.005 0.857 protein (Fragment) OS =Campylobacter jejuni subsp. jejuni 87459 GN = cje34_06680 PE = 4 SV =1 - [H8BN80_CAMJU] W8J4X8 Chemotaxis protein 72.3 36.42 189 6 20 0.8731.095 1.015 0.994 OS = Campylobacter jejuni subsp. jejuni NCTC 11168-Kf1GN = N919_00720 PE = 4 SV = 1 - [W8J4X8_CAMJE] A0A059H748 Formatedehydrogenase 82.3 12.08 36 6 7 1.311 0.622 0.795 1.077 OS =Campylobacter jejuni Cj2 GN = N215_08730 PE = 4 SV = 1 -[A0A059H748_CAMJU] A0A059GQU9 Cytochrome C 69.2 15.90 39 6 8 0.785 0.8820.892 0.817 OS = Campylobacter jejuni 255 GN = L034_04325 PE = 4 SV =1 - [A0A059GQU9_CAMJU] Q9PI32 50S ribosomal protein L7/L12 13.1 48.00 125 5 0.555 1.156 0.899 0.849 OS = Campylobacter jejuni subsp. jejuniserotype O:2 (strain NCTC 11168) GN = rp1L PE = 3 SV = 1 - [RL7_CAMJE]N4Y7C8 ATP synthase subunit alpha 52.0 20.21 26 5 9 0.943 0.985 1.0421.145 OS = Campylobacter jejuni subsp. jejuni ICDCCJ07004 GN = atpA PE =3 SV = 1 - [N4Y7C8_CAMJU] W2ANI8 Chemotaxis protein 70.2 35.36 175 5 190.557 1.240 0.923 0.655 (Fragment) OS = Campylobacter jejuni subsp.jejuni. 81-176- UMCW7 GN = X909_03500 PE = 4 SV = 1 - [W2ANI8_CAMJJ]A0A059GKB1 Uncharacterized protein 20.5 42.63 20 5 7 1.416 0.969 0.6882.228 OS = Campylobacter jejuni 30286 GN = N196_05010 PE = 4 SV = 1 -[A0A059GKB1_CAMJU] E5Z8A4 PPIC-type PHASE domain 29.4 29.66 23 5 7 1.6571.318 1.852 1.268 protein OS = Campylobacter jejuni subsp. jejuniDFVF1099 GN = CSQ_1024 PE = 4 SV = 1 - [E5Z8A4_CAMJU] E5ZDN6 Periplasmicnitrate 99.4 13.13 23 5 10 1.247 0.694 0.745 1.406 reductase, largesubunit (Fragment) OS = Campylobacter jejuni subsp. jejuni 305 GN = napAPE = 3 SV = 1 - [E5ZDN6_CAMJU] A0A059GE96 Inosine-5-monophosphate 52.123.09 28 5 8 1.060 1.048 1.203 0.976 dehydrogenase OS = Campylobacterjejuni 10186 GN = N194_07695 PE = 4 SV = 1 - [A0A059GE96_CAMJU]A0A059GIX6 Pyruvate-flavodoxin 131.3 6.32 34 5 6 1.155 0.905 0.998 1.104oxidoreductase OS = Campylobacter jejuni 10186 GN = N194_00565 PE = 4 SV= 1 - [A0A059GIX6_CAMJU] A0A059H231 DNA-directed RNA 155.5 4.29 34 5 51.143 1.013 1.208 1.014 polymerase subunit beta OS = Campylobacterjejuni Cj5 GN = N213_08515 PE = 4 SV = 1 - [A0A059H231_CAMJU] Q3HR22Putative ATP/GTP binding 33.8 23.76 33 5 6 0.551 1.156 0.934 0.700protein (Fragment) OS = Campylobacter jejuni PE = 4 SV = 1 -[Q3HR22_CAMJU] A8FNQ7 DNA-directed RNA 37.7 18.10 10 4 6 0.858 0.9930.931 1.056 polymerase subunit alpha OS = Campylobacter jejuni subsp.jejuni serotype O:6 (strain 81116/NCTC 11828) GN = rpoA PE = 3 SV = 1 -[RPOA_CAMJ8] D3FK01 Alkyl hydroperoxide reductase 22.0 36.87 6 4 5 1.1411.110 1.085 1.255 OS = Campylobacter jejuni subsp. jejuni (strainIA3902) GN = ahpC PE = 4 SV = 1 - [D3FK01_CAMJI] A0A059GHU6 Lipoprotein17.8 33.94 10 4 5 0.520 1.095 0.681 0.994 OS = Campylobacter jejuni30286 GN = N196_08005 PE = 4 SV = 1 - [A0A059GHU6_CAMJU] H8BUP1 Putativeoxidoreductase 26.0 25.64 14 4 4 1.497 1.388 1.418 1.468 subunit OS =Campylobacter jejuni subsp. jejuni 140-16 GN = cje4_08690 PE = 4 SV =1 - [H8BUP1_CAMJU] A0A059GU70 Serine protease 51.0 15.04 31 4 6 1.5481.469 1.572 1.562 OS = Campylobacter jejuni 30318 GN = N212_01200 PE = 4SV = 1 - [A0A059GU70_CAMJU] D3FN39 Bipartate energy taxis 19.3 24.85 194 4 0.375 2.172 1.256 0.740 response protein cetB OS = Campylobacterjejuni subsp. jejuni (strain IA3902) GN = cetB PE = 4 SV = 1 -[D3FN39_CAMJI] A0A059I201 Chemotaxis protein CheY 34.8 18.77 15 4 40.820 1.034 0.965 0.955 (Fragment) OS = Campylobacter jejuni K5 GN =N218_00260 PE = 4 SV = 1 - [A0A059I201_CAMJU] E5ZAL2 Ketol-acidreductoisomerase 36.7 16.27 24 3 4 0.851 1.126 1.009 1.019 OS =Campylobacter jejuni subsp. jejuni 305 GN = ilvC PE = 3 SV = 1 -[E5ZAL2_CAMJU] H7Y518 Major outer membrane 45.6 30.90 215 3 10 0.1090.832 0.840 0.121 protein OS = Campylobacter jejuni subsp. jejuni LMG23269 GN = cje114_01222 PE = 4 SV = 1 - [H7Y518_CAMJU] D6BWG0Fibronectin-binding protein 23.4 17.54 32 3 4 0.478 1.008 0.784 0.678(Fragment) OS = Campylobacter jejuni GN = cadF PE = 3 SV = 1 -[D6BWG0_CAMJU] N4Y4W1 Fumarate reductase iron-sulfur 24.9 24.77 11 3 40.737 0.776 0.716 0.868 subunit OS = Campylobacter jejuni subsp. jejuniICDCCJ07004 GN = H741_1738 PE = 4 SV = 1 - [N4Y4W1_CAMJU] A0A059HYE2 50Sribosomal protein L5 19.7 26.55 16 3 4 0.880 0.878 1.078 0.736(Fragment) OS = Campylobacter jejuni K5 GN = N218_12355 PE = 4 SV = 1 -[A0A059HYE2_CAMJU] T2D4H6 Nitrogen fixation protein NifU 32.4 19.73 22 35 1.099 1.379 1.207 1.364 OS = Campylobacter jejuni subsp. jejuni00-2544 GN = N755_00276 PE = 3 SV = 1 - [T2D4H6_CAMJU] H7X7T5 Non-hemeiron protein 17.0 48.99 17 3 6 1.198 0.956 0.748 1.957 OS =Campylobacter jejuni subsp. jejuni 51494 GN = cje10_07457 PE = 4 SV =1 - [H7X7T5_CAMJU] A0A059I263 50S ribosomal protein L13 15.7 30.50 9 3 40.784 0.864 0.928 0.765 OS = Campylobacter jejuni K5 GN = N218_00710 PE= 4 SV = 1 - [A0A059I263_CAMJU] A0A059GLY8 O-acetylhomoserine 46.6 10.6425 3 3 0.531 1.165 1.089 0.666 aminocarboxypropyltransferase OS =Campylobacter jejuni 30286 GN = N196_03230 PE = 4 SV = 1 -[A0A059GLY8_CAMJU] E5ZGX0 30S ribosomal protein S1 60.1 6.58 39 3 31.167 0.899 0.966 1.180 OS = Campylobacter jejuni subsp. jejuni 327 GN =CSU_0110 PE = 3 SV = 1 - [E5ZGX0_CAMJU] A0A059HDM4 Ferritin OS =Campylobacter 19.5 28.74 7 3 4 0.679 1.225 1.263 0.729 jejuni Cj1 GN =N214_07950 PE = 4 SV = 1 - [A0A059HDM4_CAMJU] A0A059H490 Capsulebiosynthesis protein 42.5 9.68 37 3 3 0.848 1.123 1.000 1.034 OS =Campylobacter jejuni 30318 GN = N212_06885 PE = 4 SV = 1 -[A0A059H490_CAMJU] A0A059H487 Cytochrome Cbb3 31.1 12.54 14 3 3 0.6050.898 0.814 0.799 OS = Campylobacter jejuni Cj5 GN = N213_05015 PE = 4SV = 1 - [A0A059H487_CAMJU] A0A059H9K5 Uncharacterized protein 16.124.31 21 3 3 1.324 1.130 1.047 1.527 OS = Campylobacter jejuni Cj1 GN =N214_00085 PE = 4 SV = 1 - [A0A059H9K5_CAMJU] E6RSV9 Putativeperiplasmic protein 26.4 21.03 39 3 4 0.734 1.145 1.044 0.897 OS =Campylobacter jejuni subsp. jejuni (strain S3) GN = CJS3_0034 PE = 4 SV= 1 - [E6RSV9_CAMJS] H7ZHL3 Thioredoxin 11.3 39.42 3 3 3 1.049 1.0961.010 1.208 OS = Campylobacter jejuni subsp. jejuni 2008-1025 GN =cje145_07496 PE = 3 SV = 1 - [H7ZHL3_CAMJU] A5KGI0 Trigger factor 50.99.26 32 3 4 1.196 0.959 1.622 0.690 OS = Campylobacter jejuni subsp.jejuni CG8486 GN = tig PE = 3 SV = 1 - [A5KGI0_CAMJU] A1VYF9 Acylcarrier protein 8.6 44.16 5 2 3 0.834 1.042 0.962 0.954 OS =Campylobacter jejuni subsp. jejuni serotype O:23/36 (strain 81-176) GN =acpP PE = 3 SV = 1 - [ACP_CAMJJ] A7H623 Protein RecA 37.0 8.75 13 2 20.333 0.827 1.507 0.199 OS = Campylobacter jejuni subsp. doylei (strainATCC BAA-1458/RM4099/269.97) GN = recA PE = 3 SV = 1 - [RECA_CAMJD]A7H646 50S ribosomal protein L14 13.3 15.57 4 2 2 1.392 0.788 1.1970.996 OS = Campylobacter jejuni subsp. doylei (strain ATCCBAA-1458/RM4099/269.97) GN = rplN PE = 3 SV = 1 - [RL14_CAMJD] A8FJQ050S ribosomal protein L27 9.3 32.14 4 2 2 0.427 0.991 0.987 0.466 OS =Campylobacter jejuni subsp. jejuni serotype O:6 (strain 81116/NCTC11828) GN = rpmA PE = 3 SV = 1 - [RL27_CAMJ8] A0A059GI82 Preproteintranslocase subunit 98.0 2.67 39 2 2 1.461 1.058 1.347 1.299 SecA OS =Campylobacter jejuni 10186 GN = N194_01400 PE = 4 SV = 1 -[A0A059GI82_CAMJU] R4VJ36 Methyl-accepting chemotaxis 40.5 9.89 68 2 31.356 0.726 0.954 1.120 protein (Fragment) OS = Campylobacter jejuni PE= 4 SV = 1 - [R4VJ36_CAMJU] E5ZGP1 Translation initiation factor IF-74.4 3.52 45 2 2 1.518 0.950 1.416 1.106 2 (Fragment) OS = Campylobacterjejuni subsp. jejuni 305 GN = infB PE = 3 SV = 1 - [E5ZGP1_CAMJU]A0A059HWV0 Molecular chaperone DnaK 44.2 6.80 30 2 2 1.115 1.065 1.1401.161 (Fragment) OS = Campylobacter jejuni K5 GN = dnaK PE = 4 SV = 1 -[A0A059HWV0_CAMJU] D2MY32 DNA-binding protein HU 10.3 58.16 7 2 4 0.5231.053 0.764 0.782 OS = Campylobacter jejuni subsp. jejuni 414 GN =C414_000220094 PE = 3 SV = 1 - [D2MY32_CAMJU] E5Z911 10 kDa chaperonin(Fragment) 8.5 33.33 11 2 2 0.833 1.334 1.307 0.937 OS = Campylobacterjejuni subsp. jejuni DEVF1099 GN = CSQ_1483 PE = 3 SV = 1 -[E5Z911_CAMJU] A5KF33 DNA-directed RNA 71.4 5.35 39 2 3 1.072 0.8521.198 0.829 polymerase OS = Campylobacter jejuni subsp. jejuni CG8486 GN= Cj8486_0470 PE = 3 SV = 1 - [A5KF33_CAMJU] A0A059GK15 Chemotaxisprotein CheY 25.5 10.76 11 2 2 1.082 1.172 0.861 1.497 OS =Campylobacter jejuni 30286 GN = N196_06275 PE = 4 SV = 1 -[A0A059GK15_CAMJU] H7X9T0 Putative periplasmic 10.8 26.00 9 2 3 2.1950.784 0.518 3.608 cytochrome C OS = Campylobacter jejuni subsp. jejuniLMG 23216 GN = cje100_00683 PE = 4 SV = 1 - [H7X9T0_CAMJU] H8A1L3Succinyl-CoA ligase [ADP- 41.8 10.85 31 2 4 1.598 0.731 0.879 1.443forming] subunit beta OS = Campylobacter jejuni subsp. jejuni 1997-1 GN= sucC PE = 3 SV = 1 - [H8A1L3_CAMJU] E5ZK16 Aspartate ammonia-lyase51.5 18.24 28 2 7 2.064 0.774 1.001 1.821 OS = Campylobacter jejunisubsp. jejuni 327 GN = aspA PE = 4 SV = 1 - [E5ZK16_CAMJU] H8AV03Flavodoxin 17.1 46.63 15 2 5 0.869 1.453 0.941 1.505 OS = Campylobacterjejuni subsp. jejuni 1997-11 GN = cje23_01298 PE = 3 SV = 1 -[H8AV03_CAMJU] E6S147 Heat shock protein HtpG 43.4 8.49 38 2 2 1.2110.971 1.132 1.129 OS = Campylobacter jejuni subsp. jejuni serotype HS:41(strain ICDCCJ07001) GN = ICDCCJ07001_468 PE = 4 SV = 1 - [E6S147_CAMJC]T2DA57 Biotin sulfoxide reductase 83.5 4.83 24 2 3 1.297 0.947 1.3940.957 OS = Campylobacter jejuni subsp. jejuni 00-2538 GN = N565_00245 PE= 3 SV = 1 - [T2DA57_CAMJU] W2AJL6 Hemolysin D 35.9 7.29 29 2 2 0.9231.075 1.067 1.011 OS = Campylobacter jejuni subsp. jejuni 81-176-DRH212GN = X908_07140 PE = 4 SV = 1 - [W2AJL6_CAMJJ] A0A059HWP5 50S ribosomalprotein L6 19.6 13.48 11 2 2 0.685 0.939 0.944 0.740 OS = Campylobacterjejuni 20176 GN = N195_02450 PE = 4 SV = 1 - [A0A059HWP5_CAMJU]A0A059I3A1 Bifunctional aconitate 68.5 7.91 41 2 4 1.215 1.015 0.9601.395 hydratase 2/2-methylisocitrate dehydratase (Fragment) OS =Campylobacter jejuni K5 GN = N218_00085 PE = 4 SV = 1 -[A0A059I3A1_CAMJU] E5Z763 Histidine-binding protein 16.6 12.00 25 2 20.536 1.228 0.916 0.781 OS = Campylobacter jejuni subsp. jejuni DFVF1099GN = CSQ_0818 PE = 4 SV = 1 - [E5Z763_CAMJU] N4Y795 Periplasmic nitratereductase, 18.5 23.08 14 2 2 1.045 0.800 0.679 1.337 electron transfersubunit OS = Campylobacter jejuni subsp. jejuni ICDCCJ07004 GN =H741_0553 PE = 3 SV = 1 - [N4Y795_CAMJU] E5ZDN8 Major antigenic peptidePEB2 23.4 11.06 24 2 2 1.820 0.919 1.187 1.624 OS = Campylobacter jejunisubsp. jejuni 305 GN = CSS_1183 PE = 4 SV = 1 - [E5ZDN8_CAMJU]A0A023WJ10 Multifunctional 53.7 7.87 41 2 3 2.246 0.792 1.851 1.044aminopeptidase A OS = Campylobacter jejuni subsp. jejuni CG8421 GN =CJ8421_04595 PE = 4 SV = 1 - [A0A023WJ10_CAMJU] E5Z8E0Ubiquinol-cytochrome c 17.0 26.75 18 2 3 0.661 1.010 0.843 0.861reductase, iron-sulfur subunit OS = Campylobacter jejuni subsp. jejuniDFVF1099 GN = petA PE = 4 SV = 1 - [E5Z8E0_CAMJU] A3ZFB9 Protein TolB(Fragment) 37.1 7.51 20 2 2 1.168 0.974 1.030 1.200 OS = Campylobacterjejuni subsp. jejuni HB93-13 GN = CJJHB9313_0128 PE = 4 SV = 1 -[A3ZFB9_CAMJU] E5ZCI8 Cytochrome c oxidase, cbb3- 21.0 15.14 11 2 20.654 0.917 0.830 0.766 type, subunit II (Fragment) OS = Campylobacterjejuni subsp. jejuni 305 GN = ccoO PE = 4 SV = 1 - [E5ZCI8_CAMJU] E5ZDB1UDP-N-acetylglucosamine 1- 43.3 6.47 29 2 2 1.339 0.913 1.188 1.119carboxyvinyltransferase (Fragment) OS = Campylobacter jejuni subsp.jejuni 305 GN = murA PE = 3 SV = 1 - [E5ZDB1_CAMJU] A8FP13 50S ribosomalprotein L29 7.0 11.48 4 1 1 OS = Campylobacter jejuni subsp. jejuniserotype O:6 (strain 81116/NCTC 11828) GN = rpmC PE = 3 SV = 1 -[RL29_CAMJ8] A7H1N3 50S ribosomal protein L31 7.3 15.38 4 1 1 1.0750.960 1.247 0.899 OS = Campylobacter jejuni subsp. doylei (strain ATCCBAA-1458/RM4099/269.97) GN = rpmE PE = 3 SV = 1 - [RL31_CAMJD] E7G1N630S ribosomal protein S12 13.4 13.93 6 1 2 1.278 0.572 0.758 1.051 OS =Campylobacter jejuni subsp. jejuni DFVF1099 GN = rpsL PE = 3 SV = 1 -[E7G1N6_CAMJU] E5ZKB7 Methyl-accepting chemotaxis 66.0 3.42 77 1 2 1.4191.018 1.069 1.467 protein (MCP) signaling domain protein OS =Campylobacter jejuni subsp. jejuni 327 GN = CSU_1383 PE = 4 SV = 1 -[E5ZKB7_CAMJU] H7Y9X9 50S ribosomal protein L25 19.5 12.36 8 1 2 OS =Campylobacter jejuni subsp. jejuni 55037 GN = rplY PE = 3 SV = 1 -[H7Y9X9_CAMJU] E5Z7S7 HAD-superfamily hydrolase, 114.6 1.54 2 1 1 1.2750.867 1.201 1.000 subfamily IA, variant 1 family protein OS =Campylobacter jejuni subsp. jejuni DFVF1099 GN = CSQ_1071 PE = 4 SV =1 - [E5Z7S7_CAMJU] A0A059HMD1 Membrane protein 45.7 23.29 114 1 8 0.2240.963 0.840 0.279 OS = Campylobacter jejuni Cj2 GN = N215_00405 PE = 4SV = 1 - [A0A059HMD1_CAMJU] A0A059GML6 Thiamine biosynthesis 47.4 2.3329 1 1 1.462 0.787 1.142 1.095 protein ThiC OS = Campylobacter jejuni255 GN = L034_06270 PE = 4 SV = 1 - [A0A059GML6_CAMJU] A0A059GQ49 NADHdehydrogenase 24.9 6.57 8 1 1 0.793 0.891 0.886 0.866 OS = Campylobacterjejuni 10186 GN = N194_04245 PE = 4 SV = 1 - [A0A059GQ49_CAMJU] D2MZ94Fibronectin type III domain 45.2 3.74 26 1 2 protein OS = Campylobacterjejuni subsp. jejuni 414 GN = C414_000260115 PE = 4 SV = 1 -[D2MZ94_CAMJU] A0A059HT33 30S ribosomal protein S6 12.5 6.60 9 1 1 0.8571.009 1.187 0.792 (Fragment) OS = Campylobacter jejuni K5 GN =N218_16365 PE = 4 SV = 1 - [A0A059HT33_CAMJU] A0A059HR90 Superoxidedismutase 24.8 14.09 15 1 3 OS = Campylobacter jejuni K1 GN = N217_00445PE = 4 SV = 1 - [A0A059HR90_CAMJU] H7ZK03 ATP-dependent chaperone 31.67.39 49 1 2 1.019 1.107 1.339 0.915 protein ClpB (Fragment) OS =Campylobacter jejuni subsp. jejuni 2008-894 GN = cje146_02596 PE = 4 SV= 1 - [H7ZK03_CAMJU] I6YES0 Flagellin A (Fragment) 58.9 22.24 355 1 100.477 0.897 0.908 0.512 OS = Campylobacter jejuni GN = flaA PE = 4 SV =1 - [I6YES0_CAMJU] H7QVG3 DNA-binding response 34.4 2.03 2 1 1 3.8110.308 1.290 0.990 regulator, putative OS = Campylobacter coli 111- 3 GN= cco1_05089 PE = 4 SV = 1 - [H7QVG3_CAMCO] A0A059HJZ0 Uncharacterizedprotein 13.9 13.22 9 1 1 1.048 0.911 1.037 1.000 OS = Campylobacterjejuni Cj2 GN = N215_00975 PE = 4 SV = 1 - [A0A059HJZ0_CAMJU] A3YTA0Ribosomal protein L3 18.6 10.00 9 1 2 0.614 0.838 0.685 0.816 (Fragment)OS = Campylobacter jejuni subsp. jejuni 260.94 GN = rplC PE = 4 SV = 1 -[A3YTA0_CAMJU] A3ZGX1 Flagellin subunit protein 59.2 19.06 241 1 8 2.8320.535 0.869 1.896 FlaB OS = Campylobacter jejuni subsp. jejuni 84-25 GN= flaB PE = 4 SV = 1 - [A3ZGX1_CAMJU] H7QW70 RNA polymerase sigma factor72.7 1.13 16 1 1 1.195 1.023 1.175 1.130 RpoD OS = Campylobacter coli111-3 GN = rpoD PE = 3 SV = 1 - [H7QW70_CAMCO] A0A059GGJ8 Aspartateammonia-lyase 51.7 19.23 11 1 8 2.534 0.638 0.947 1.855 OS =Campylobacter jejuni 10186 GN = aspA PE = 4 SV = 1 - [A0A059GGJ8_CAMJU]A0A059GN06 50S ribosomal protein L1 25.0 10.30 4 1 2 0.724 1.063 0.7901.058 OS = Campylobacter jejuni 255 GN = L034_06405 PE = 4 SV = 1 -[A0A059GN06_CAMJU] A0A023WJ77 2-oxoglutarate-acceptor 41.1 10.96 20 1 21.317 1.251 1.456 1.230 oxidoreductase subunit OorA OS = Campylobacterjejuni subsp. jejuni CG8421 GN = oorA PE = 4 SV = 1 - [A0A023WJ77_CAMJU]H7XU40 Putative transmembrane 30.7 9.71 32 1 3 protein OS =Campylobacter jejuni subsp. jejuni 60004 GN = cje11_00070 PE = 4 SV =1 - [H7XU40_CAMJU] H7XPE7 Uncharacterized protein 28.2 2.81 27 1 1 0.6021.000 0.949 0.689 OS = Campylobacter jejuni subsp. jejuni LMG 23263 GN =cje109_01311 PE = 4 SV = 1 - [H7XPE7_CAMJU] H7YV89 Uncharacterizedprotein 14.7 7.20 27 1 1 0.825 0.963 0.972 0.888 (Fragment) OS =Campylobacter jejuni subsp. jejuni ATCC 33560 GN = cje135_02523 PE = 4SV = 1 - [H7YV89_CAMJU] A0A059HSX2 Membrane protein 17.7 6.04 11 1 10.672 1.062 0.884 0.877 OS = Campylobacter jejuni 20176 GN = N195_04100PE = 4 SV = 1 - [A0A059HSX2_CAMJU] A5KI22 ATP synthase F1 sector 27.712.81 22 1 3 0.666 1.217 0.853 1.033 gamma subunit OS = Campylobacterjejuni subsp. jejuni. CG8486 GN = Cj8486_0101 PE = 4 SV = 1 -[A5KI22_CAMJU] A0A059I444 Branched-chain amino acid 32.7 6.80 18 1 2aminotransferase (Fragment) OS = Campylobacter jejuni K5 GN = N218_11890PE = 4 SV = 1 - [A0A059I444_CAMJU] E5ZGJ6 Delta-aminolevulinic acid 34.32.89 27 1 1 dehydratase OS = Campylobacter jejuni subsp. jejuni 305 GN =CSS_2245 PE = 3 SV = 1 - [E5ZGJ6_CAMJU] E5ZFV6 Fibronectin type IIIdomain 28.2 8.40 43 1 2 2.266 0.906 0.963 2.318 protein (Fragment) OS =Campylobacter jejuni subsp. jejuni 305 GN = CSS_1989 PE = 4 SV = 1 -[E5ZFV6_CAMJU] A0A059GNV7 2-oxoglutarate ferredoxin 31.2 4.27 14 1 11.156 1.022 1.005 1.276 oxidoreductase subunit beta OS = Campylobacterjejuni 10186 GN = N194_05915 PE = 4 SV = 1 - [A0A059GNV7_CAMJU] A5KFW1Membrane bound zinc 40.7 7.55 20 1 2 1.000 0.979 1.086 0.979metallopeptidase OS = Campylobacter jejuni subsp. jejuni CG8486 GN =Cj8486_1154c PE = 3 SV = 1 - [A5KFW1_CAMJU] H8A911 Putative endonuclease64.2 1.65 9 1 1 (Fragment) OS = Campylobacter jejuni subsp. jejuni2008-979 GN = cje160_10022 PE = 4 SV = 1 - [H8A911_CAMJU] X0N637 30Sribosomal protein S15 10.0 10.23 4 1 1 0.180 0.759 1.075 0.138 OS =Campylobacter jejuni X GN = rpsO PE = 3 SV = 1 - [X0N637_CAMJU] Q29XU550S ribosomal protein L15 11.4 10.58 13 1 1 0.464 1.015 0.568 0.901(Fragment) OS = Campylobacter jejuni GN = rpsLO PE = 3 SV = 1 -[Q29XU5_CAMJU] A0A059HZ15 30S ribosomal protein S5 13.3 9.45 4 1 1 0.3741.290 0.833 0.629 (Fragment) OS = Campylobacter jejuni K5 GN =N218_07570 PE = 4 SV = 1 - [A0A059HZ15_CAMJU] A0A059GK29 Uncharacterizedprotein 21.0 14.21 15 1 2 OS = Campylobacter jejuni 30286 GN =N196_05955 PE = 4 SV = 1 - [A0A059GK29_CAMJU] E1PQD5 Transcriptiontermination 38.8 3.16 11 1 1 0.856 1.519 1.405 1.005 factor Rho OS =Campylobacter jejuni subsp. jejuni serotype HS21 (strain M1/99/308) GN =rho PE = 3 SV = 1 - [E1PQD5_CAMJM] A0A059GFH4 7-alpha-hydroxysteroid28.1 8.49 23 1 2 1.528 0.847 1.582 0.888 dehydrogenase OS =Campylobacter jejuni 255 GN = L034_05345 PE = 4 SV = 1 -[A0A059GFH4_CAMJU] W2AH04 50S ribosomal protein L21 10.4 10.53 9 1 10.584 1.000 0.737 0.861 OS = Campylobacter jejuni subsp. jejuni81-176-DRH212 GN = X908_06720 PE = 3 SV = 1 - [W2AH04_CAMJJ] D2MWN2Uncharacterized protein 8.9 11.84 2 1 1 1.634 1.000 1.134 1.566 OS =Campylobacter jejuni subsp. jejuni 414 GN = 0414_000040068 PE = 4 SV =1 - [D2MWN2_CAMJU] A0A059H8K1 Ubiquinol cytochrome C 48.1 2.40 10 1 10.689 0.984 0.821 0.897 oxidoreductase OS = Campylobacter jejuni Cj1 GN= N214_01665 PE = 4 SV = 1 - [A0A059H8K1_CAMJU] W2U6P5 Uncharacterizedprotein 5.8 19.23 22 1 1 OS = Campylobacter jejuni subsp. jejuni81-176-UMCW9 GN = X910_08590 PE = 4 SV = 1 - [W2U6P5_CAMJJ] E5ZLJ73-dehydroquinate dehydratase 17.2 6.41 8 1 1 1.063 0.892 1.114 0.838 OS= Campylobacter jejuni subsp. jejuni 327 GN = aroQ PE = 3 SV = 1 -[E5ZLJ7_CAMJU] A3YNF7 Oxaloacetate decarboxylase, 65.8 2.50 16 1 1 1.6730.966 1.200 1.463 alpha subunit, putative OS = Campylobacter jejunisubsp. jejuni CF93-6 GN = CJJCF936_1007 PE = 4 SV = 1 - [A3YNF7_CAMJU]Q29VV8 Putative nucleotidyl sugar 28.5 8.27 15 1 2 epimerase OS =Campylobacter jejuni subsp. jejuni serotype O:23/36 (strain 81-176) GN =CJB1426c PE = 4 SV = 1 - [Q29VV8_CAMJJ] A0A059I1M1 30S ribosomal proteinS7 15.0 9.02 5 1 1 0.704 1.048 0.835 0.961 (Fragment) OS = Campylobacterjejuni K5 GN = N218_01130 PE = 4 SV = 1 - [A0A059I1M1_CAMJU] W2AGH3 Furfamily transcriptional 8.8 13.92 7 1 1 1.333 1.021 1.255 1.178 regulatorOS = Campylobacter jejuni subsp. jejuni 81-176- DRH212 GN = X908_06930PE = 4 SV = 1 - [W2AGH3_CAMJJ] E5ZEC9 Methionyl-tRNA synthetase 9.713.41 45 1 1 2.302 0.862 1.589 1.380 (Fragment) OS = Campylobacterjejuni subsp. jejuni 305 GN = CSS_1425 PE = 4 SV = 1 - [E5ZEC9_CAMJU]Q001V3 Putative cytochrome C-type 17.3 7.33 13 1 1 0.902 1.006 1.0380.951 haem-binding periplasmic protein (Fragment) OS = Campylobacterjejuni PE = 4 SV = 1 - [Q001V3_CAMJU] E5ZBV9 30S ribosomal protein S86.0 22.64 8 1 1 0.718 0.827 0.960 0.672 (Fragment) OS = Campylobacterjejuni subsp. jejuni 305 GN = CSS_0466 PE = 4 SV = 1 - [E5ZBV9_CAMJU]A5KH24 Glutamate-1-semialdehyde 2,1- 44.3 2.69 27 1 1 1.252 0.933 0.9211.378 aminomutase OS = Campylobacter jejuni subsp. jejuni CG8486 GN =hemL PE = 1 SV = 1 - [A5KH24_CAMJU] A0A023WHY9 Succinyl-CoA synthase,alpha 30.0 9.69 15 1 2 1.069 0.851 0.923 1.071 subunit OS =Campylobacter jejuni subsp. jejuni CG8421 GN = CJ8421_02610 PE = 4 SV =1 - [A0A023WHY9_CAMJU] A0A023WJZ2 Bifunctional adhesin/ABC 28.2 4.25 251 1 1.215 0.799 0.747 1.412 transporter aspartate/glutamate-bindingprotein OS = Campylobacter jejuni subsp. jejuni CG8421 GN = CJ8421_04555PE = 4 SV = 1 - [A0A023WJZ2_CAMJU] E5ZG55 Nucleoside diphosphate kinase11.4 10.68 11 1 1 OS = Campylobacter jejuni subsp. jejuni 305 GN =CSS_2085 PE = 3 SV = 1 - [E5ZG55_CAMJU] A0A059HYD1 Enolase (Fragment)36.6 3.53 33 1 1 OS = Campylobacter jejuni K5 GN = eno PE = 4 SV = 1 -[A0A059HYD1_CAMJU] A3YPW8 Uncharacterized protein 13.1 10.17 18 1 1(Fragment) OS = Campylobacter jejuni subsp. jejuni 260.94 GN =CH26094_1432 PE = 4 SV = 1 - [A3YPW8_CAMJU] A0A059GQ16 50S ribosomalprotein L16 16.3 8.51 4 1 1 OS = Campylobacter jejuni 10186 GN =N194_05010 PE = 4 SV = 1 - [A0A059GQ16_CAMJU] E6RY61 Cytochrome cbiogenesis 43.7 3.45 41 1 1 1.247 1.052 1.234 1.154 protein,CcmF/CycK/CcsA family OS = Campylobacter jejuni subsp. jejuni serotypeHS:41 (strain ICDCCJ07001) GN = ICDCCJ07001_974 PE = 4 SV = 1 -[E6RY61_CAMJC] A5KHZ1 Uncharacterized protein 33.8 4.03 24 1 1 2.0390.831 0.889 2.072 OS = Campylobacter jejuni subsp. jejuni CG8486 GN =Cj8486_0065 PE = 4 SV = 1 - [A5KHZ1_CAMJU] A0A059GQV0 Flagellar basalbody protein 19.9 6.18 11 1 1 0.539 1.269 0.875 0.850 FliL OS =Campylobacter jejuni 255 GN = L034_04560 PE = 4 SV = 1 -[A0A059GQV0_CAMJU] E5ZIW7 Elongation factor P 20.5 7.07 8 1 1 OS =Campylobacter jejuni subsp. jejuni 327 GN = efp PE = 3 SV = 1 -[E5ZIW7_CAMJU] A1VX93 Membrane protein, putative, 10.8 13.98 9 1 1 0.4781.120 1.284 0.453 degenerate OS = Campylobacter jejuni subsp. jejuniserotype O:23/36 (strain 81-176) GN = CJJ81176_0041 PE = 4 SV = 1 -[A1VX93_CAMJJ] A0A059GDI2 Membrane protein 35.6 8.33 12 1 2 0.808 1.4320.862 1.459 OS = Campylobacter jejuni 30286 GN = N196_02850 PE = 4 SV =1 - [A0A059GDI2_CAMJU] A1VXK7 Uncharacterized protein 33.7 8.74 20 1 2OS = Campylobacter jejuni subsp. jejuni serotype O:23/36 (strain 81-176)GN = CJJ81176_0159 PE = 4 SV = 1 - [A1VXK7_CAMJJ] E5ZAK6Glucosamine-fructose-6- 25.0 5.88 31 1 1 1.599 1.108 1.171 1.644phosphate aminotransferase OS = Campylobacter jejuni subsp. jejuni 305GN = CSS_0017 PE = 4 SV = 1 - [E5ZAK6_CAMJU] E5ZDP4 Amino-acid ABCtransporter 22.0 6.74 22 1 1 1.000 1.001 0.965 1.127 ATP-binding proteinYecC (Fragment) OS = Campylobacter jejuni subsp. jejuni 305 GN =CSS_1189 PE = 3 SV = 1 - [E5ZDP4_CAMJU] E5ZBA2 50S ribosomal protein L418.6 8.77 17 1 1 (BL4) (Fragment) OS = Campylobacter jejuni subsp.jejuni 305 GN = CSS_0286 PE = 4 SV = 1 - [E5ZBA2_CAMJU] Q9R5T9 PEB2 =MAJOR antigenic 3.7 42.86 1 1 1 peptide (Fragment) OS = Campylobacterjejuni PE = 1 SV = 1 - [Q9R5T9_CAMJU] E5ZIC4 4-oxalocrotonatetautomerase 7.5 20.59 4 1 1 family enzyme family protein OS =Campylobacter jejuni subsp. jejuni 327 GN = dmpI PE = 4 SV = 1 -[E5ZIC4_CAMJU] A0A059GHF9 Protease OS = Campylobacter 48.0 3.85 23 1 1jejuni 10186 GN = N194_01570 PE = 4 SV = 1 - [A0A059GHF9_CAMJU]A0A059HXA8 50S ribosomal protein L2 26.5 7.05 15 1 1 0.841 0.997 0.8841.031 (Fragment) OS = Campylobacter jejuni K5 GN = N218_11745 PE = 4 SV= 1 - [A0A059HXA8_CAMJU] A0A023WM76 Homoserine O- 34.2 5.12 11 1 1 1.0230.983 1.241 0.880 succinyltransferase OS = Campylobacter jejuni subsp.jejuni CG8421 GN = CJ8421_08685 PE = 4 SV = 1 - [A0A023WM76_CAMJU]E5ZF74 Uncharacterized protein 14.5 12.10 14 1 1 1.093 0.936 1.113 0.998(Fragment) OS = Campylobacter jejuni subsp. jejuni 305 GN = CSS_1726 PE= 4 SV = 1 - [E5ZF74_CAMJU] H7YSG7 Quinone-reactive Ni/Fe- 31.9 6.19 251 1 0.635 1.131 0.790 0.989 hydrogenase, small subunit (Fragment) OS =Campylobacter jejuni subsp. jejuni LMG 23357 GN = cje133_06525 PE = 4 SV= 1 - [H7YSG7_CAMJU] H7YGU5 L-lactate permease 56.2 3.80 22 1 1 0.4941.037 0.781 0.713 (Fragment) OS = Campylobacter jejuni subsp. jejuni LMG9879 GN = cje120_05071 PE = 4 SV = 1 - [H7YGU5_CAMJU] A0A059GH71Uncharacterized protein 25.9 7.79 13 1 1 0.977 1.155 1.268 0.967 OS =Campylobacter jejuni 30286 GN = N196_08385 PE = 4 SV = 1 -[A0A059GH71_CAMJU] E5ZBY8 Plasminogen-binding protein 29.3 7.25 31 1 11.100 0.994 1.094 1.086 PgbB domain protein (Fragment) OS =Campylobacter jejuni subsp. jejuni 305 GN = CSS_0530 PE = 4 SV = 1 -[E5ZBY8_CAMJU] U4NW65 Flagellin OS = Campylobacter 59.0 21.85 350 0 10jejuni 4031 GN = BN867_13230 PE = 4 SV = 1 - [U4NW65_CAMJU]

The results in Table 1 show significant changes in protein expressionfor 34 proteins when Campylobacter jejuni NCTC 11168 wildtype or theCampylobacter jejuni 11168 T268G mutant are treated with FeQ (see ratiosof WT+FeQ/WT and MOMP-T+FeQ/MOMP-T). The impact of mutating the MOMPprotein of Campylobacter jejuni at Thr-268 to glycine is alsodemonstrated by the ratio of Campylobacter jejuni 11168 wildtype toCampylobacter jejuni 11168 T268G mutant (WT/MOMP-T in Table 1), andshowed an up-regulation with a ratio higher than 1.5 for nine proteins(Putative periplasmic cytochrome C, Aspartate ammonia-lyase,Multifunctional aminopeptidase, Succinyl-CoA ligase, DNA-bindingresponse regulator, Flagellin subunit protein FlaB, Aspartateammonia-lyase, Fibronectin type III domain protein andGlucosamine-fructose-6-phosphate aminotransferase) while ten proteinsshowed down-regulation with a ratio of less than 0.6 (Nickel-dependenthydrogenase, Putative amino-acid transporter, Chemotaxis protein(Fragment), Putative ATP/GTP binding protein, Lipoprotein, Bipartateenergy taxis response protein cetB, Major outer membrane protein,Membrane protein, Flagellin A, Membrane protein and L-lactate permease).These results at least demonstrate that glycosylation of Campylobacterjejuni 11168 wildtype affects the expression of MOMP and Flagellin A/B,which are involved in bacterial motility, adhesion to BgAgs, aggregationand biofilm formation.

Comparison of the ratios of WT+FeQ/WT in Table 1 shows that fiveproteins (Putative GMC oxidoreductase, Chemotaxis protein (Fragment),Putative amino-acid transporter, Bipartate energy taxis response proteincetB and Ferritin) were all up-regulated above 20% and ten proteins((Formate dehydrogenase, Periplasmic nitrate reductase, Putativeperiplasmic cytochrome C, Succinyl-CoA ligase, Aspartate ammonia-lyase,Multifunctional aminopeptidase A, Thiamine biosynthesis protein ThiC,DNA-binding response regulator, Aspartate ammonia-lyase (differentsubunit) and Flagellin B, FlaB) were down-regulated more than 20%.Notably, the results demonstrate that the glycosylated MOMP of thewildtype strain was down-regulated about 20%, and Flagellin B (majorsubunit of flagellum) was down-regulated about 45% when Campylobacterjejuni 11168 wildtype was treated with FeQ. These results at leastdemonstrate that FeQ has an impact on the expression of two essentialproteins involved in colonization, adhesion and motility.

Example 16. Glycosylated Campylobacter Dominates Colonization of Chickenin a Mixed Glycosylated and Non-Glycosylated Population of Campylobacter

Materials and Methods

Chickens were colonized by a mixed strain (10⁵ cfu, 50/50) ofCampylobacter jejuni 11168 wildtype (O-glycosylated) and theMOMP^(T268G) mutant of Campylobacter (non-glycosylated). The chickenswere orally challenged and after 7 days post-infection caecal sampleswere analyzed.

Results

FIG. 16 is a graph showing the colonization of chickens by a mixedstrain (10⁵ cfu, 50/50) of Campylobacter jejuni 11168 wildtype(O-glycosylated) and the MOMP^(T268G) mutant of Campylobacter(non-glycosylated). The chickens were orally challenged and after 7 dayspost-infection caecal samples were analyzed. The results show that the0-glycosylated wildtype strain dominates, and is able to establish aninfection. In contrast, the non-glycosylated strain (MOMP^(T268G)) wasunable to colonize the chicken's GI tract, and no mutant strain could bedetected.

Example 17. FeQ Prevents Formation of Biofilm on Human Teeth

Materials and Methods

Molar teeth were extracted from human patients, and mouth swabs takenfrom each patient to obtain samples of each patient's bacterial florapresent in the mouth. The mouth swabs were cultured in the laboratoryusing LB media in order to grow bacterial populations ordinarily presentin the mouth of each patient. The extracted teeth were washed andbrushed extensively using PBS buffer and ethanol. Each patient's toothwas then placed in the bacterial culture prepared from that patient'sbacterial flora sample, and cultured aerobically for 24 hours in LBmedia. The teeth were then stained with CEPLAC™ (Manx Healthcare Ltd,Warwick, UK), and washed three times with PBS-Tween (50 mL) to determineif biofilm had been formed on the teeth.

Results

All teeth stained red indicating the presence of biofilm on the teethafter just 24 hours. The same teeth were then cleaned using PBS bufferand ethanol, and the procedure repeated except with 48 hours ofculturing in the presence of FeQ (340 μM). After 48 hours, no teethstained red demonstrating that biofilms could not be established on theteeth in the presence of FeQ.

Example 18. Efficacy of FeQ and FeTyr to Reduce Campylobacter Carriagein Chickens and Promote Growth in Chickens

Materials and Methods

A study was performed to evaluate growth promotion and reduction ofCampylobacter carriage using FeQ and FeTyr in Ross 308 male broilerswith 7 treatment groups. Each treatment group comprised four replicatesof 10 birds per pen (40 birds/treatment group and 4 pens of 10birds/treatment group), and there were 2 control groups and 5 testgroups. All the test groups and one of the control groups were exposedat day 20 of the trial to dirty litter, which tested positive forCampylobacter. This method was used to provide a more natural method toCampylobacter challenge the birds. Thus there was a positive controlwhere one treatment group was challenged with Campylobacter and onenegative control group where the birds were not challenged, and fivetreatment groups that were all challenged with Campylobacter. The totalnumber of birds used in the 7 treatment groups was 280. Details of thetreatments are provided in Table 2. Treatment group 1 was a negativecontrol where birds just received the commercial feed, and were notchallenged with dirty litter containing Campylobacter. Treatment group 2was the positive control where the birds received the commercial feed,and were challenged with dirty litter containing Campylobacter at day20. Treatment group 3 received 0.22 g/L of FeQ in their drinking waterand 0.22 g/Kg FeQ in their feed during the entire trial, and waschallenged with dirty litter containing Campylobacter at day 20.Treatment group 5 received 0.22 g/L of FeQ in their drinking waterduring the entire trial, and was challenged with dirty litter containingCampylobacter at day 20. Treatment group 6 received 0.22 g/kg FeQ intheir feed during the entire trial, and was challenged with dirty littercontaining Campylobacter at day 20. Treatment group 7 received 0.022 g/LFeQ in their drinking water during the entire trial, and was challengedwith dirty litter containing Campylobacter at day 20. Treatment group 8received 0.02 g/L FeTyr in their drinking water during the entire trial,and was challenged with dirty litter containing Campylobacter at day 20.The FeTyr was pre-dissolved in DMSO, and diluted to provide a solutionof 0.02 g/L of FeTyr in water. (An additional treatment group 4 wasterminated due to solubility issues.)

TABLE 2 Treatment Details Campylobacter Treatment Description Challenge1 Control-1 Commercial feed No 2 Control-2 Commercial feed Yes 3 0.22g/L FeQ in water + 0.22 g/kg FeQ in Yes feed 5 0.22 g/L FeQ in water Yes6 0.22 g/kg FeQ in feed Yes 7 0.022 g/L FeQ in water Yes 8 0.02 g/LFeTyr in water Yes

The birds were fed with a commercial three-phase feeding program usingstarter, grower and finisher feeds with formulations shown in Table 3.All diets had coccidiostat (MAXIBAN® at 0.0625% in starter and finisherphase diets and MONTEBAN® at 0.06% in finisher phase). Xylanase(RONOZYME® WX at 200 g per ton) and phytase (RONOZYME® P at 150 gramsper ton) were added to all diets.

TABLE 3 Basal feed formulation for starter, grower and finisher dietsRaw Material STARTER % GROWER % FINISHER % Barley 10.5 8.4 7.2 Wheat50.0 55.0 60.0 Soya Ext Hipro 26.0 23.0 19.0 Full fat Soya Cherwell 5.05.0 5.0 L Lysine HCl 0.40 0.30 0.30 DL-methionine 0.40 0.35 0.30L-threonine 0.15 0.15 0.15 Soya Oil 4.0 4.50 4.75 Limestone 1.25 1.251.25 MonoCal phosphate 1.50 1.25 1.25 Salt 0.25 0.25 0.25 Sodiumbicarbarbonate 0.15 0.15 0.15 Broiler Premix 0.40 0.40 0.40 NutrientAnalysis Analysis Analysis Fat (ether extract) 6.34 6.85 7.11 Protein21.85 20.64 19.14 Fibre 3.08 3.02 2.97 Ash 6.01 5.68 5.50 ME-P 12.7813.04 13.22 Total lysine 1.45 1.28 1.17 Available lysine 1.35 1.19 1.09Methionine 0.69 0.62 0.55 Total methionine and 1.03 0.95 0.85 cysteineThreonine 0.91 0.86 0.79 Tryptophan 0.25 0.23 0.21 Calcium 0.95 0.910.89 Phosphorus 0.72 0.66 0.65 Available phosphorus 0.48 0.42 0.42 Salt0.30 0.30 0.30 Sodium 0.17 0.17 0.17 Vit A 13.20 13.5 13.50 Vit D3 5.05.0 5.00 Vit E 100 100 100

The feeding program is show in Table 4. The birds were reared in floorpens to day 42, and fed starter, grower and finisher feed at day 0 to11, 11 to 24, and 24 to 42 days, respectively. All birds were weighedindividually and feed weigh backs recorded per pen at day 0, 11, 21, 24and 42 days.

TABLE 4 Feeding Program Feeding Phase Starter Grower Finisher (days ofage) 0-11 11-24 24-42

Prior to challenging the chickens with dirty litter containingCampylobacter at day 20, each pen was tested for Campylobacter usingcloacal swabs. All pens tested negative for Campylobacter prior to thechallenge. At day 20, litter, which was naturallyCampylobacter-contaminated was tested to confirm the presence ofCampylobacter, and then added (approximately 2 kg/pen) to the litter inall pens except in pens for treatment group 1 (the negative control). Atday 28, the pen litter was sampled to confirm the presence or absence ofCampylobacter. At day 41 and 42, caecal samples were taken from 3 birdsper pen (12 birds per treatment group) and tested for Campylobacterenumeration. At day 42, digesta, fecal samples, and caecal content wastaken from all birds, and pooled per pen. Two birds per pen were alsotaken from treatment groups 1-3, euthanized, and blood samples taken.Samples were analyzed for blood chemistry, including analysis foralkaline phosphatase, aspartate amino transferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, totalprotein, albumin, globulin, amylase and glucose.

In order to minimize risk of cross-contamination, standard industrybiosecurity measures were used including: disinfecting boots, changingovershoes and gloves between pens/treatments, entering Campylobacternegative pens before entering Campylobacter positive pens, and leavingadjacent pens empty. Daily health, culls, and mortality were recorded.All bird weights were recorded at 0, 11, 21, 24, 33 and 42 days. Weightgains, feed intake and feed conversion ratio (FCR) were derived for eachfeeding period.

The effect of the treatment groups compared to the negative controlgroup (treatment group 1) and the positive control group (treatmentgroup 2) is shown in Tables 5-12 for the periods 0-11 days, 11-20 days,20-25 days, 11-25 days, 25-42 days, 20-42 days, 0-20 days, and 0-42days.

Results

FIG. 17 shows the average body weight at day 42 for all treatmentgroups, and a comparison to a commercial control labeled “Target”. Thefigure shows that treatment group 1 (the negative control labeled “CNC”)attained an average body weight (ABW) of 3.437 kg at day 42 (which washigher than the commercial target of 2.979 kg). The positive control(labeled “CC”), which was challenged with dirty litter containingCampylobacter at day 20, in contrast only attained an ABW of 3.186 kg atday 42, which was significantly less than the negative control(treatment group 1). This result demonstrates that challenging withdirty litter contaminated with Campylobacter resulted in a reduction ofgrowth of the chicken by an average of 251 grams. However, when thechickens were challenged with dirty litter containing Campylobacter buttreated with FeQ or FeTyr in treatment groups 3, 5, 6, 7 and 8, alltreatment groups performed better than the positive controldemonstrating that FeQ and FeTyr treatment had a positive effect ongrowth. In fact, FeQ in feed at 0.22 g/kg (treatment group 6) producedchicken with an ABW of 3.464 kg, which was higher than the negativecontrol ABW of 3.437 kg even though treatment group 6 had beenchallenged with dirty litter containing Campylobacter.

FIG. 18 shows the mortality adjusted feed conversion rate (MFCR) at day42 for all treatment groups, and a comparison to a commercial controllabeled “Target”. (A lower MFCR number is a better result.) The figureshows that treatment group 1 (the negative control labeled “CNC”) had aMFCR of 1.563, which was lower than the commercial target of 1.703. Thepositive control, labeled “CC” which was challenged with the dirtylitter containing Campylobacter at day 20 had a significantly higherMFCR of 1.679 than the negative control. Thus challenging with dirtylitter infected with Campylobacter resulted in a higher MFCR. However,when the chickens were challenged with dirty litter infected withCampylobacter but treated with FeQ or FeTyr in treatment groups 3, 5, 6,7 and 8, all treatment groups performed better than the positive controldemonstrating that FeQ and FeTyr treatment had a positive effect on MFCR(i.e. decreasing the numerical MFCR). The results show that treatmentgroups 3, 5, 6, 7 and 8 had MFCR values of 1.595, 1.560, 1.563, 1.612and 1.577, respectively. Furthermore, treatment groups 5 and 6 performedas well as the negative control even when challenged with dirty littercontaining Campylobacter.

FIG. 19 shows the number of Campylobacter colony forming units per gram(cfu/g) of bird droppings at day 42 for treatment groups 1-3 and 6-8. (Alower number is a better result.) The results show that treatment groups3 and 6-8 all performed better than the positive control (treatmentgroup 2) demonstrating that FeQ and FeTyr had a positive effect onreducing Campylobacter infection of poultry. Notably, chicken treatedwith FeTyr, FeQ in feed, and FeQ in feed and water all had colonyforming units of Campylobacter per gram of dropping that were similarto, or less than, those of the negative control group (treatment group1). The detection of low levels of Campylobacter in the negativecontrols demonstrates how highly contagious the bacterium is, and islikely to be an indication that a small number of birds in the negativecontrol group became infected despite not being experimentallychallenged with dirty litter. The results in FIG. 19 for the lowconcentration of FeQ in water (0.022 g/L; treatment group 7) appears toshow less of an effect than the other treatment groups, although thisdifference was considered more likely due to experimental error forexample following cross contamination of samples. As discussed below,the results obtained from a further experiment, as given in FIG. 20confirm that treatment group 7 did, indeed, also provide the highlybeneficial effect.

FIG. 20 shows the average number of Campylobacter colony forming unitsper gram (cfu/g) of caeca samples at day 42 for treatment groups 1-3 and5-8. The results show that all the treatment groups (3 and 5-8) allperformed better than the positive control (treatment group 2)demonstrating that FeQ and FeTyr had a positive effect on reducingCampylobacter infection of poultry.

The effect of the treatments on overall liveability and Europeanproduction and efficiency factor (EPEF) is shown in Table 5.(EPEF=[(Liveability×Live weight in kg at end of trial/Age in Days×FCRcommercial)×100].

The effect of FeQ treatment on growth performance in the absence ofCampylobacter challenge during the starter phase (0-11 days) and periodfrom 0-20 days is shown in Table 14. Since the negative and positivecontrols (treatment groups 1 and 2) are identical prior to challengewith the dirty litter at day 20, these groups may be pooled forcomparison to treatment groups 3, 5, 6, and 7 in order to see if FeQ hadan effect on growth in the absence of a challenge by dirty littercontaminated with Campylobacter during the first 20 days of growth. Theresults demonstrate that FeQ promotes growth of chicken even in theabsence of a challenge from dirty litter contaminated withCampylobacter. At day 20, the average body weight (ABW) for the controlgroups (treatment groups 1 and 2) is 0.927 kg versus 0.963 kg fortreatment groups 3, 5, 6 and 7 which all received FeQ. This improvementin body weight is also reflected in a significantly better MFCR for theFeQ treated birds. Table 11 shows the MFCR for the birds treated ingroups 3, 5, 6 and 7 is 1.2996 versus 1.3374 for the control groups(treatment groups 1 and 2). Notably the P-value is less than 0.05.

The same positive effect of FeTyr treatment on growth performance in theabsence of Campylobacter challenge is also evident from Table 11. TheAWG during the first 20 days of production for chicken treated withFeTyr (treatment group 8) is 0.895 kg compared to 0.884 and 0.889 kg fortreatment groups 1 and 2 (negative and positive controls). Furthermore,the MFCR during the first 20 days of production for chicken treated withFeTyr (treatment group 8) is 1.311 versus 1.32 and 1.355 for treatmentgroups 1 and 2, respectively. (A lower MFCR value is an improvement.)

The results of this study demonstrate that both FeQ and FeTyr promotegrowth and decrease the mortality adjusted feed conversion ratio (MFCR)in the absence or presence of dirty litter contaminated withCampylobacter.

TABLE 5 Effect of treatments on growth performance during starter phase(day 0-11) ABW AFD AWG MFCR Treatment Day 0 Day 11 Day 0-11 1 0.0400.331 0.348 0.291 1.239^(b) 2 0.040 0.337 0.359 0.297 1.228^(b) 3 0.0400.346 0.356 0.306 1.181^(ab) 5 0.040 0.334 0.352 0.294 1.210^(ab) 60.041 0.351 0.360 0.310 1.168^(a) 7 0.040 0.325 0.348 0.285 1.236^(b) 80.040 0.329 0.353 0.289 1.229^(b) P-value 0.136 0.418 0.979 0.463 0.005SED 0.000 0.013 0.016 0.013 0.018 P-value for contrast 1 vs 2 0.5120.667 0.519 0.682 0.584 1 vs 2 to 8 0.666 0.573 0.603 0.583 0.045 2 vs3567 0.632 0.844 0.723 0.834 0.054 5 vs 6 0.099 0.213 0.627 0.233 0.0335 vs 7 0.141 0.466 0.804 0.494 0.170 2 vs 8 0.645 0.538 0.709 0.5490.982 ^(a-b)within a column reflects differences between treatments whenP < 0.05; SED = Standard errors of difference of means; ABW = averagebody weight (kg); AFD = average feed intake (kg); AWG = average weightgain (kg); MFCR = Mortality adjusted feed conversion ratio; FCR = Feedconversion ratio-commercial.

TABLE 6 Effect of treatments on growth performance during grower phase(day 11-20) ABW AFD AWG MFCR Treatment Day 20 Day 11-20 1 0.924 0.7990.593 1.362 2 0.929 0.838 0.592 1.421 3 0.972 0.857 0.625 1.375 5 0.9430.821 0.609 1.348 6 0.991 0.841 0.640 1.343 7 0.947 0.829 0.622 1.333 80.935 0.809 0.606 1.351 P-value 0.358 0.311 0.279 0.279 SED 0.032 0.0250.021 0.036 P-value for contrast 1 vs 2 0.875 0.133 0.977 0.115 1 vs 2to 8 0.248 0.094 0.175 0.987 2 vs 3567 0.189 0.961 0.075 0.020 5 vs 60.145 0.427 0.160 0.884 5 vs 7 0.913 0.737 0.546 0.673 2 vs 8 0.8500.253 0.516 0.065 SED = Standard errors of difference of means; ABW =average body weight (kg); AFD = average feed intake (kg); AWG = averageweight gain (kg); MFCR = Mortality adjusted FCR; FCR = FCR commercial.

TABLE 7 Effect of treatments on growth performance during period day20-25. ABW AFD AWG MFCR Treatment Day 25 Day 20-25 1 1.366 0.662 0.4421.500 2 1.371 0.652 0.442 1.550 3 1.424 0.667 0.453 1.477 5 1.384 0.6580.441 1.495 6 1.426 0.685 0.434 1.599 7 1.388 0.661 0.441 1.513 8 1.3770.662 0.442 1.499 P-value 0.723 0.916 0.999 0.882 SED 0.044 0.026 0.0300.096 P-value for contrast 1 vs 2 0.912 0.685 0.998 0.604 1 vs 2 to 80.403 0.932 0.996 0.759 2 vs 3567 0.339 0.444 0.990 0.707 5 vs 6 0.3610.311 0.826 0.294 5 vs 7 0.930 0.902 0.988 0.854 2 vs 8 0.892 0.6930.999 0.604 SED = Standard errors of difference of means; ABW = averagebody weight (kg); AFD = average feed intake (kg); AWG = average weightgain (kg); MFCR = Mortality adjusted FCR; FCR = FCR commercial.

TABLE 8 Effect of treatments on overall growth performance during growerphase (day 11-25) AFD AWG MFCR Treatment Day 11-25 1 1.462 1.035 1.421 21.490 1.034 1.457 3 1.524 1.078 1.417 5 1.479 1.050 1.409 6 1.526 1.0751.440 7 1.490 1.064 1.406 8 1.471 1.048 1.414 P-value 0.660 0.804 0.598SED 0.042 0.036 0.030 P-value for contrast 1 vs 2 0.516 0.984 0.241 1 vs2 to 8 0.293 0.406 0.891 2 vs 3567 0.657 0.267 0.118 5 vs 6 0.280 0.4980.300 5 vs 7 0.787 0.707 0.925 2 vs 8 0.664 0.695 0.165 SED = Standarderrors of difference of means; ABW = average body weight (kg); AFD =average feed intake (kg); AWG = average weight gain (kg); MFCR =Mortality adjusted FCR.

TABLE 9 Effect of treatments on overall growth performance duringfinisher phase (day 25-42) ABW AFD AWG MFCR Treatment Day 42 Day 25-42 13.437 3.479 2.070^(b) 1.688 2 3.186 3.480 1.814^(a) 1.889 3 3.342 3.3871.918^(ab) 1.773 5 3.407 3.357 2.023^(b) 1.706 6 3.464 3.315 2.039^(b)1.704 7 3.304 3.362 1.916^(ab) 1.793 8 3.341 3.434 1.964^(ab) 1.716P-value 0.027 0.56 0.009 0.211 SED 0.075 0.099 0.062 0.081 P-value forcontrast 1 vs 2 0.004 0.997 <.001 0.022 1 vs 2 to 8 0.110 0.247 0.0160.233 2 vs 3567 0.004 0.129 0.004 0.035 5 vs 6 0.455 0.680 0.800 0.988 5vs 7 0.187 0.960 0.101 0.294 2 vs 8 0.053 0.649 0.027 0.046 ^(a-b)withina column reflects differences between treatments when P < 0.05; SED =Standard errors of difference of means; ABW = average body weight (kg);AFD = average feed intake (kg); AWG = average weight gain (kg); MFCR =Mortality adjusted FCR; FCR = FCR commercial.

TABLE 10 Effect of treatments on the growth performance during theexperimental period of day 20-42 (after the birds were challenged) AFDAWG MFCR Treatment Day 20-42 1 4.142 2.512^(b) 1.653 2 4.131 2.256^(a)1.820 3 4.054 2.370^(ab) 1.713 5 4.015 2.464^(ab) 1.665 6 4.0012.473^(ab) 1.678 7 4.023 2.357^(ab) 1.739 8 4.096 2.406^(ab) 1.676P-value 0.767 0.025 0.344 SED 0.110 0.068 0.075 P-value for contrast 1vs 2 0.926 0.001 0.038 1 vs 2 to 8 0.306 0.028 0.290 2 vs 3567 0.2290.008 0.055 5 vs 6 0.898 0.894 0.856 5 vs 7 0.941 0.138 0.331 2 vs 80.752 0.042 0.070 ^(a-b)within a column reflects differences betweentreatments when P < 0.05; SED = Standard errors of difference of means;ABW = average body weight (kg); AFD = average feed intake (kg); AWG =average weight gain (kg); MFCR = Mortality adjusted.

TABLE 11 Overall effect of treatments on growth performance during theexperimental period of day 0-20 (before birds were challenged). AFD AWGMFCR Treatment Day 0-20 1 1.147 0.884 1.320 2 1.196 0.889 1.355 3 1.2130.931 1.310 5 1.173 0.903 1.303 6 1.201 0.951 1.284 7 1.178 0.907 1.3028 1.161 0.895 1.311 P-value 0.623 0.368 0.225 SED 0.038 0.032 0.025P-value for contrast 1 vs 2 0.215 0.881 0.181 1 vs 2 to 8 0.191 0.2510.627 2 vs 3567 0.860 0.188 0.012 5 vs 6 0.476 0.150 0.469 5 vs 7 0.9070.899 0.978 2 vs 8 0.371 0.846 0.094 SED = Standard errors of differenceof means; ABW = average body weight (kg); AFD = average feed intake(kg); AWG = average weight gain (kg); MFCR = Mortality adjusted.

TABLE 12 Overall effect of treatment groups on growth performance (day0-42) AFD AWG MFCR Treatment Day 0-42 1 5.289 3.397^(b) 1.563 2 5.3283.145^(a) 1.679 3 5.267 3.302^(ab) 1.595 5 5.188 3.367^(ab) 1.560 65.201 3.423^(b) 1.563 7 5.201 3.265^(ab) 1.612 8 5.258 3.301^(ab) 1.577P-value 0.920 0.028 0.193 SED 0.132 0.075 0.047 P-value for contrast 1vs 2 0.773 0.004 0.024 1 vs 2 to 8 0.633 0.111 0.352 2 vs 3567 0.290.004 0.018 5 vs 6 0.920 0.461 0.954 5 vs 7 0.924 0.190 0.284 2 vs 80.601 0.053 0.043 ^(a-b)within a column reflects differences betweentreatments when P < 0.05; SED = Standard errors of difference of means;ABW = average body weight (kg); AFD = average feed intake (kg); AWG =average weight gain (kg); MFCR = Mortality adjusted.

TABLE 13 The effect of treatments on overall liveability and Europeanproduction and efficiency factor (EPEF) EPEF Treatment Day 20 Day 42 1318.3 282.8 2 334.7 250.7 3 350.4 262.9 5 352.0 278.3 6 364.8 265.0 7354.5 276.2 8 336.4 296.0 P-value 0.547 0.842 SED 23.83 31.68 P-valuefor contrast 1 vs 2 0.500 0.323 1 vs 2 to 8 0.111 0.645 2 vs 3567 0.2850.437 5 vs 6 0.599 0.680 5 vs 7 0.919 0.949 2 vs 8 0.945 0.170

TABLE 14 Effect of treatments on growth performance in absence ofCampylobacter challenge during starter phase (0-11 days) and period 0-20days. ABW ABW AFD 0-20 AWG 0-20 Treatment Day 11 Day 20 days days MFCR0-20 Groups 1 & 0.334 0.927 1.172 0.887 1.3374 2 FeQ 0.339 0.963 1.1910.923 1.2996 (Groups 3, 5, 6, 7) P-value 0.584 0.079 0.432 0.078 0.029SED 0.009 0.020 0.024 0.020 0.016 ABW = average body weight (kg); AFD =average feed intake (kg); AWG = average weight gain (kg); MFCR =mortality adjusted feed conversion ratio

Example 19. FeDOPA Treatment Makes Antibiotic Resistant Strain ofEnteropathogenic E. coli (EPEC) E2348/69 Lose Resistance to Antibiotic

Materials and Methods

The impact on the growth curve of antibiotic resistant EnteropathogenicE. coli (EPEC) strain E2348/69 (genotype Wild Type EPEC O17:H6) whengrown in the presence of a fixed concentration of gentamicin (1.25 μM)and an increasing concentration of FeDOPA versus the strain grown in thepresence of only FeDOPA or only gentamicin, was determined.

Results

FIGS. 21A-C are graphs that show the impact on the growth curve ofantibiotic resistant Enteropathogenic E. coli (EPEC) strain E2348/69(genotype Wild Type EPEC O17:H6) when grown in the presence of a fixedconcentration of gentamicin (1.25 μM) and an increasing concentration ofFeDOPA (FIG. 21A: 130 μM, FIG. 21B: 160 μM and FIG. 11C: 200 μM) versusthe strain grown in the presence of only FeDOPA or only gentamicin. Thegraphs show that the rate of growth of the strain was inhibited in thepresence of gentamicin and FeDOPA relative to the rate of growth of thestrain just in the presence of gentamicin. This is evidence that FeDOPAcan be used in conjunction with antibiotics to kill or inhibit thegrowth of antibiotic resistant bacteria.

Example 20. FeDOPA Prevents Attachment of Bacteria to Surfaces

Materials and Methods

Enteropathogenic E. coli (EPEC) E2348/69 were grown in wells for 48hours at 37° C. in the presence of FeDOPA (10-250 μM), and in theabsence of FeDOPA (as control). After 48 hours, the wells were washed inorder to remove suspended cells. Crystal violet was then added to eachwell. The wells were then washed to remove excess dye. A mixture ofacetone/ethanol was then added to the wells to re-suspend any cellsattached to the plastic surface of the wells, and dissolve any dyepresent. The presence of dye in each well was then quantified bymeasuring the O.D. at 570 nm.

Results

In the absence of FeDOPA, EPEC binds to the plastic surface and forms abiofilm that is readily detected by dying with crystal violet. However,in the presence of FeDOPA, EPEC attachment to the plastic surface andformation of a biofilm is inhibited. FIG. 22 shows quantitatively thedifference in the attachment of EPEC cells to the plastic well surfacein the absence and presence of FeDOPA by measurement of the opticalabsorbance of crystal violet that was absorbed by EPEC cells attached tothe surface. At an FeDOPA concentration of 68-250 μM attachment ofbacterial cells to the surface and biofilm formation is inhibited.

Example 21. Campylobacter jejuni Loses Motility after Treatment with FeQ

Campylobacter jejuni NCTC 11168 was treated with FeQ (34 μM) and a platecontaining brain-heart infusion (BHI) medium inoculated with 5 μl, 2×10⁵colony forming units of the treated bacteria and the plate was culturedfor 43 hours. The growth and motility of the bacteria after treatmentwith FeQ was compared to a positive control where the bacteria had notbeen treated with FeQ, and also to a negative control where no bacteriawere applied to a plate of the BHI medium. The data (not shown) showedthat after 43 hours, the Campylobacter jejuni treated with FeQ had aclear zone around the bacteria indicating that the bacteria was notmotile. In contrast, Campylobacter jejuni that was not treated with FeQwas motile, and spread around the culture plate. There was no growthvisible, as expected, on the plates that were not inoculated withbacteria. The experiment demonstrates that the Campylobacter jejuniloses motility after treatment with FeQ, and is consistent with theresults obtained by iTRAQ analysis that demonstrate that FeQ downregulates FlaA expression (the Flagella of Campylobacter).

Example 22. Disruption of a Preformed Biofilm with FeTyr

Crystal violet assays were used as described above to demonstrate thatFeTyr could disrupt a pre-formed biofilm. A mature biofilm formed byEPEC-pgA⁺⁺ was treated with FeTyr for 24, 48 and 72 hours at FeTyrconcentrations of 100 μM, 150 μM and 200 μM and the presence of thebiofilm after these times was compared to an untreated biofilm (labeled“Control”) using a crystal violet assay. The color of the control wellswas more intense in color at 72 hours than those that were treated withFeTyr at 100, 150 and 200 μM for 72 hours. FIG. 23 shows quantitativelythe optical absorbance of crystal violet at 570 nm that was absorbed bythe EPEC cells that remained attached to the surface of the plastic wellafter a mature biofilm formed by EPEC-pgA⁺⁺ was treated with FeTyr(shown as FeY in FIG. 23) at 100 μM, 150 μM and 200 μM compared to anuntreated biofilm (labeled “Control”) in the crystal violet assay. Asignificantly lower optical absorbance was found at 72 hours for thebiofilm treated with FeTyr at 100, 150 and 200 μM at 72 hours. Theseresults demonstrate that FeTyr can disrupt a pre-formed biofilm.

Example 23. Disruption of a Preformed Biofilm with FeTyr and Fe-DOPA

A BioFlux system was used to demonstrate that FeTyr and Fe-DOPA can beused to disrupt a mature EPEC-ΔcsrA biofilm. Our studies showed thatthat a mature biofilm of EPEC-ΔcsrA can be formed in the presence of LBmedium 30% v/v and imaged (data not shown). The mature biofilm wastreated with FeTyr at concentrations of 100, 150 and 200 μM for 20 hoursand compared to a control biofilm that had just been treated with LBmedium 30% v/v. It was found that biofilm dispersion increased as theconcentration of FeTyr was increased from 100 to 200 μM (data notshown). Mature biofilm was treated with FeDOPA at a concentration of 100μM for 20 hours and compared to a control biofilm that had just beentreated with LB medium 30% v/v. It was found that Fe-DOPA dispersed thebiofilm at a concentration of 100 μM (data not shown).

Example 24. Treatment of an Acne Patient with FeQ

An 18 year old female patient was treated continuously for 30 days byapplying a solution of ferric quinate (340 μM) to her acne vulgarism(“acne”) once each day. Within 5 days of the start of treatment, heracne, which had not previously responded to treatment with antibiotics,began to show signs of healing. After treatment for 30 days, her acnewas completely healed. Her acne did not recur even after discontinuationof treatment for over one year.

Example 25. Effect of FeQ on Biofilm Formation of a Medical Device

Materials and Methods

To investigate the effect of Ferric Quinate (FeQ) on the surfaceintegrity of contact lenses, two contact lenses were independentlyincubated in either saline solution, or saline solution with a finalconcentration of 340 μM FeQ at 4° C. for 7 days, whilst gently shaking.The lenses were then washed 6 times with phosphate-buffered solution(PBS)+0.05% Tween. Each lens was then washed twice with distilled waterbefore analysis via environmental scanning electron microscope (ESEM).

To investigate biofilm formation on the contact lens, clinicallydetermined PAO-1 strains of Pseudomonas aeruginosa were incubated withthe lenses in either Luria-Bertani Media (LB) or LB with a finalconcentration of 340 μM FeQ at 37° C. for 24 hours in a non-shakingincubator. The lenses were then washed 6 times with PBS+0.05% Tween,before being stored overnight in PBS+0.05% Tween. In preparation for theESEM, formaldehyde was added to a final concentration of 1% andincubated for 10 minutes in order to inactivate the bacteria. The lenseswere then washed 4 times with PBS+0.05% Tween, and immediately beforeanalysis the lenses were washed a further two times with distilledwater.

Results

Surface Integrity

Lenses were treated with 340 μM FeQ or left untreated (control) toinvestigate the effect, if any, of FeQ treatment on the surfaceintegrity of the contact lens.

The results show that FeQ at 340 μM does not have any visiblesignificant effect (via ESEM) on the surface integrity of the contactlens.

Biofilm Formation

ESEM images (data not shown) following incubation with bacteria onlyshowed large scale biofilm development of Pseudomonas aeruginosa, overthe surface of the contact lens. Analysis via the ESEM requires vacuumand causes areas of the biofilm to dehydrate, which is responsible forthe perforated appearance of the biofilm of the contact lens. Thedehydration gives perception of depth, and shows the biofilm formed inthe absence of FeQ to be substantial. Individual bacteria were visiblein the biofilm, surrounded by the extracellular matrix (ECM).

The impact of incubation with bacteria in the presence of FeQ 340 μM wasalso investigated. These images (data not shown) showed that, in thepresence of FeQ at 340 μM, Pseudomonas aeruginosa appears as eithersingle bacterium, or small aggregates of bacteria, with no apparent ECMformation. These results indicate that FeQ inhibits biofilm formation ofPseudomonas aeruginosa.

Based on these results, it can be concluded the FeQ and other compoundsas described herein can be used to inhibit or prevent biofilm formationon medical devices, such as but not limited to, contact lenses.

Example 26. Metabolomic Analysis

Materials and Methods

Strains analysed were the wild type Campylobacter jejuni NCTC 11168, anda mutant (MOMP268T/G) where Thr-268 of the MOMP protein of the wild typeis mutated to glycine, resulting in a strain of the bacteria in whichthe MOMP protein cannot be glycosylated. The mutant is further describedin WO 2013/121214. The strains were grown for 48 hours in Mueller HintonBroth (MHB). In non-control samples, the bacteria (wild type and mutant)were treated with FeQ at a concentration of 340 μM.

Three replicates of a sample were taken from each culture, and eachreplicate was analysed three times (i.e. producing 9 reads for eachsample). Thus, for example, the sample taken from fresh media (FM)produced nine reads, labelled FM-1_1, 1_2, 1_3, 2_1, 2_2, 2_3, 3_1, 3_2and 3_3, respectively. It is the same for each of the other samples,which are:

-   -   SMWT: spent media from the wild type control culture    -   SMWTF: spent media from the wild type culture grown in the        presence of FeQ    -   SMMT: spent media from the mutant control culture    -   SMMTF: spent media from the mutant culture grown in the presence        of FeQ

For metabolite profiling, LC was performed on an Accela system (ThermoFisher Scientific, Hemel Hempstead, UK). Chromatographic separation wascarried out using a ZIC-pHILIC (150 mm×4.6 mm, 5 μm column, MerckSequant) as previously described (Creek et al. 2011, Anal Chem 83,8703-8710). Briefly, the column was maintained at 45° C. and sampleswere eluted with a linear gradient from 80% B to 5% B over 15 min,followed by an 2 min linear gradient from 5% B to 80% B, and 7 minre-equilibration with 80% B at the flow rate of 300 μl/min. Mobile phaseA was 20 mM ammonium carbonate in water and mobile phase B wasacetonitrile 100% acetonitrile. The injection volume was 10 μl andsamples were maintained at 4° C. An Orbitrap Exactive (Thermo FisherScientific, Hemel Hempstead, UK) with a HESI 2 probe was operated inpolarity switching mode, with the following settings: resolution 50 000,AGC 1×10⁶, m/z range 70-1400, sheath gas 40, auxiliary gas 5, sweep gas1, probe temperature 150° C., and capillary temperature 275° C. Forpositive mode ionisation: source voltage +4.5 kV, capillary voltage +50V, tube voltage +70 kV, skimmer voltage +20 V. For negative modeionisation: source voltage −3.5 kV, capillary voltage −50 V, tubevoltage −70 V, skimmer voltage −20 V. Mass calibration was performed foreach polarity immediately before each analysis batch. The calibrationmass range was extended to cover small metabolites by inclusion oflow-mass contaminants with the standard Thermo calibration mixturemasses (below m/z 1400), C₂H₆NO₂ for positive ion electrosprayionisation (PIESI) mode (m/z 76.0393) and C₃H₅O₃ for negative ionelectrospray ionisation (NIESI) mode (m/z 89.0244).

Data Processing and Analysis

Raw LC-MS data were processed with XCMS for untargeted peak-picking(Tautenhahn et al. 2008, BMC Bioinformatics 9, 504) and mzMatch.R forpeak matching and annotation of related peaks (Scheltema et al. 2011,Analytical Chemistry 83, 2786-2793). Putative metabolite identificationwas carried out by IDEOM using the default parameters (Creek et al.2012, Analytical Chemistry 84, 8442-8447). Metabolite identification wasperformed by matching accurate masses and retention times of authenticstandards (Level 1 metabolite identification according to theMetabolomics Standards Initiative (Sumner et al. 2014, Metabolomics 10,1047-1049; Sumner et al. 2007, Metabolomics 3, 211-221). However, whenstandards were not available, predicted retention times were used, hencethese identifications should be considered as putative (Level 2identification).

Results and Conclusions

FIG. 24A shows the data from positive mode analysis, as an OPLS-DAscores plot. This shows a clear separation between fresh media (FM) andother spent media (SMWT; SMWTF; SMMT; SMMTF) which demonstrates that alot of metabolites were excreted and consumed during cell culture.

FIG. 24B also shows the data from the positive mode analysis. The freshmedia (FM) results were removed from the plot, because they are sodifferent from the other samples so that any differences between thedifferent spent media samples could be hidden. This plot shows a clearseparation between wild type (SMWT) and wild type+FeQ (SMMTF), but theSMMT and SMMTF clustered more closely. This indicates that FeQ does notcause large detectable change between mutant and mutant+FeQ. Overall, itis clear that there are less metabolic changes caused by FeQ in themutant than in the wild type.

FIG. 24C contrasts from FIG. 24A in that it shows the data from thenegative mode analysis, although essentially the same pattern andconclusions apply as in FIG. 24A.

FIG. 24D contrasts with FIG. 24B in that it shows the data from thenegative mode analysis. The negative mode data in FIG. 24D showsslightly different trends from the positive mode data, and demonstratesa clear separation between mutant (SMMT) and mutant+FeQ (SMMTF) samples,as well as between the SMWT and SMWTF samples.

These data demonstrate how fundamentally the metabolism of bacteria ischanged by treatment with FeQ. This is consistent with the phenotypicchanges observed in bacteria treated with FeQ (as confirmed by iTRAQresults as discussed in Example 15), and provides an insight into themechanism underlying the ability of FeQ and its related compounds asdiscussed in section III.A of this application to treat bacteria andcause an inhibition in their ability to form biofilm, colonise chickensand other animals, and even make the bacteria less resistance toantibiotics.

Example 27. Preparation Protocol for K[Fe(C₇H₁₁O₆)₃](OH)₃H₂O (FeQ)

FeCl₃.6H₂O (50 g, 184 mmol, Alfa Aesar, 97%) was placed in a flask anddissolved in 300 mL of H₂O (J.T. baker, HPLC grade). To that solution,D-(−)-quinic acid (110 g, 572 mmol, Buchlr Gmbh, 96%) was added slowlywith continuous stirring. The pH of the solution was adjusted to ˜3 byaddition of 10M KOH (Alfa Aesar, 85%) (˜80 mL was required).

The dark yellow solution darkened to brownish upon addition of KOH. Thedark solution was stirred for 1 h at room temperature. After stirring atroom temperature for 1 h ethanol (EMD, 94%) (2.5 l) was added slowly tothe solution with stirring.

After addition of approximately of the total ethanol, the solutionlightened visibly and a fine solid began to precipitate from solution.After addition of the remaining ethanol, the solution is allowed to sitovernight at room temperature.

The solids are collected by vacuum filtration on a fritted funnel andallowed to dry on the funnel while the vacuum is continued for 2-3 h.The bright yellow solid is spread in a thin layer in a drying dish anddried open to the air for 3 days followed by drying under vacuum for 48h to give 155 g of the final product.

Example 28. Synthesis of Fe(Tyr)₃

L-tyrosine (5.43 g, 30 mmol, Chem Impex, 99.5%) and LiOH.H₂O (1.26 g, 30mmol, EMD, 94%) were dissolved in water (250 ml, J.T. Baker, HPLCgrade)) and the solution heated to 70° C. for 20 min. The FeCl₃ salt(1.62 g, 10 mmol, Alfa Aesar, 98%)) was dissolved in a minimum quantityof water (3-5 ml) and was added to the tyrosine/LiOH solution.

Precipitation (brown solid) was almost instantaneous but stirring withheating continued for 15 min. The product was allowed to cool to roomtemperature and was collected by filtration. The product was air driedand then further dried in a lyophilizer. Isolated yield was 5.85 g.

Example 29. Synthesis of Fe(DOPA)₃

L-Dopa (11.84 g, 60 mmol, AK Scientific, 98%) and LiOH.H₂O (2.52 g, 60mmol, EMD, 94%) were dissolved in water (100 ml, J.T. Baker, HPLC grade)and the solution heated to 70° C. for 20 min. The FeCl₃ salt (3.2 g, 20mmol, Alfa Aesar, 98%) was dissolved in a minimum quantity of water(6-10 ml) and was added to the Dopa/LiOH solution vigorous.

Precipitation (very dark purple) was almost instantaneous but stirringwith heating continued for 15 min.

The product was allowed to cool to room temperature and was collected byfiltration. The product was air dried and then further dried in alyophilizer. Isolated yield was 6.5 g. More solid (4 g) was collectedfrom the filtrate and dried in the same way. Overall yield was 10.5 g.

Example 30. Fe-Q and Fe-Phe Potentiate the Effect of Antibiotics

Methods

To investigate effects upon antibiotic resistance, a laboratory strainof Psuedomonas aeruginosa (PAO1N) and a mixed population of clinicalisolates (PAO Mixed) were incubated in Luria-Bertani (LB) media alone,or with different concentrations (34 μM, 100 μM, 200 μM and 340 μM) ofFeQ or FePhe.

Each of the different media, bar one control, contained 10 μg/ml of theaminoglycoside antibiotic Amikacin.

10 μl of the bacterial strains were added into each well of a 96-wellmicro-titer plate, before 290 μl of the relevant media was added towells. Each different condition was repeated in sextuplicate.

The plate was incubated at 37° C. within a micro-titer plate reader for17.5 hours, with the OD₆₀₀ read every 30 minutes.

Results

The results are shown in FIGS. 25A and 25B. These figures show that Fe-Qand Fe-Phe provide similar effects at reducing tolerance of PAO1N andPAO Mixed to the aminoglycoside Amikacin.

We claim:
 1. A method of treating microbial infection/colonization in ananimal in an amount effective to enhance growth and weight gain, themethod comprising administering to the infected or at risk animal apharmaceutical or veterinary product, a medical device or a dietaryproduct comprising one or more compounds having the structure:

wherein X, X¹, and X² are NH₂, and R, R¹, and R² are independentlyselected from the group consisting of H, CH₃, CH₂SH, CH₂CO₂H,CH₂CH₂CO₂H, CH₂C₆H₅, CH(CH₃)CH₂CH₃, CH₂CH(CH₃)₂, CH₂CH₂SCH₃, CH₂CH₂CH₂,CH₂OH, CH(OH)CH₃, CH(CH₃)₂, or CH₂C₆H₄OH, wherein R′, R^(1′), and R^(2′)are H; wherein the compound has the formula

and the compound is formed of iron complexed with alpha-amino acids,wherein the alpha-amino acid is selected from the group consisting ofalanine, aspartic acid, cysteine, methionine, glutamic acid, glycine,isoleucine, leucine, phenylalanine, proline, serine, threonine,tyrosine, and valine, wherein the alpha-amino acid is L-form, D-form ora mixture of D, L-forms, wherein the compound is the only ferric aminoacid complex in the pharmaceutical, veterinary, or dietary product ormedical device.
 2. The method of claim 1, wherein the animal is selectedfrom the group consisting of poultry, chicken, turkey, ducks; livestock,cattle, sheep, goats, swine, fish, shellfish, domestic animal, human andaquatic animal.
 3. The method of claim 2, wherein the animal is achicken selected from the group consisting of broiler chicken, a pullet,hen, and a breeder chicken.
 4. The method of claim 3, further comprisingrearing the animal for a period of time less than 47, 46, 45, 44, 43,42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21 or 20 days to permit enhanced growth.
 5. The method ofclaim 2, wherein the animal is chicken, fed a total amount of chickenfeed that is reduced from a mean average of 123.8 g feed per day, by0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%,15%, 20%, 25% or more, during its rearing.
 6. The method of claim 1,wherein the enhanced growth of an animal includes enhancing one or morecharacteristics selected from the group consisting of enhancing bodyweight or average body weight (ABW), feed intake or average feed intake(AFD), weight gain average weight gain (AWG), and mortality adjustedfeed conversion ratio (MFCR).
 7. The method of claim 1, wherein thepharmaceutical or veterinary product, medical device or dietary productis administered to the animal separately, simultaneously, orsequentially with the administration of one or more antimicrobialagents.
 8. The method of claim 7, wherein the animal is infected with anantibiotic-resistant microorganism selected from the group consisting ofa gram positive bacterium, a gram negative bacterium, a biofilm-formingbacterium, Streptococcus pneumoniae, Campylobacter, Neisseriagonorrhoeae, Salmonella, Methicillin-resistant Staphylococcus aureus(MRSA), Shigella, Vancomycin-resistant Enterococcus (VRE),Vancomycin-resistant Staphylococcus aureus (VRSA),Erythromycin-resistant Group A Streptococcus, Clindamycin-resistantGroup B Streptococcus, Carbapenem-resistant Enterobacteriaceae (CRE),drug-resistant tuberculosis, Extended spectrum Enterobacteriaceae(ESBL), multidrug-resistant Acinetobacter (including MRAB), Clostridiumdifficile, Enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa, H.pylori, Streptococcus anginosus and Uropathogenic E. coli (UPEC), S.epidermidis, E. faecalis, E. coli, S. aureus, Campylobacter, andPseudomonas or combinations thereof.
 9. The method of claim 7, whereinthe amount of the one or more antimicrobial agents is less than atherapeutically effective or therapeutically optimal dose of the one ormore antimicrobial agents when administered to the patient or animalthat is not in receipt of the product.
 10. The method of claim 7,wherein the one or more antimicrobial agent is an antibiotic selectedfrom the group consisting of aminoglycosides, ansaycins, carbapenems,cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides,monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides,quinolones/fluoroquinolone, sulfonamides, tetracyclines, clofazimine,dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid,pyrazinamide, rifampicin (rifampin), rifabutin, rifapentine,streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid,metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin,thiamphenicol, tigecycline, tinidazole, and trimethoprim; andcombinations thereof.
 11. The method of claim 7, wherein the one or morecompounds is administered to a subject by parenteral delivery; enteraldelivery; oral delivery; topical delivery; buccal delivery; dermaldelivery; transdermal delivery; sublabial delivery; sublingual delivery;in combination with dental product selected from the group consisting oftoothpaste, a mouthwash, a mouth shield, a dental instrument, dentures,dental retainers, braces a dental floss, toothpicks, bristles oftoothbrushes, dental prostheses and orthodontic devices; or incombination with chewable non-food items selected from the groupconsisting of foods, toys, dog bones and dog biscuits.
 12. The method ofclaim 8, wherein the biofilm is associated with a bacterial infectionselected from the group consisting of impetigo, boils, abscesses,folliculitis, cellulitis, necrotizing fasciitis, pyomyositis,surgical/traumatic wound infection, and infected ulcers and burns,osteomyelitis, device-related osteoarticular infections, impetigo,secondarily infected skin lesions, meningitis, brain abscess, subduralempyema, spinal epidural abscess, arterial damage, gastritis, urinarytract infections, biliary tract infections, pyelonephritis, cystitis,sinus infections, ear infections, otitis media, otitis externa, leprosy,tuberculosis, conjunctivitis, bloodstream infections, benign prostatichyperplasia, chronic prostatitis, lung infections, osteomyelitis,catheter infections, bloodstream infections, skin infections, acne,rosacea, dental caries, periodontitis, gingivitis, nosocomialinfections, arterial damage, endocarditis, periprosthetic jointinfections, open or chronic wound infections, venous stasis ulcers,diabetic ulcers, arterial leg ulcers, pressure ulcers, endocarditis,pneumonia, orthopedic prosthesis and orthopedic implant infections,peritoneal dialysis peritonitis, cirrhosis, and other acute or chronicinfection that involves or possesses a biofilm.
 13. The method of claim12, wherein the biofilm is associated with cystic fibrosis.